Method and materials for detecting Legionella pneumophila

ABSTRACT

The invention provides novel genes and proteins of Legionela pneumophila. The invention also provides methods of detecting or quantitating L. pneumophila using these genes, mRNAs encoded by the genes, or proteins encoded by the genes as targets. Nucleic acids designed to hybridize with the genes or mRNAs encoded by the genes, or antibodies that bind specifically to the proteins, are used in the methods, and the nucleic acids and antibodies can be provided in kits.

This invention was made, in part, under Public Health Service GrantsAI30064 and AI34937 from the National Institutes of Health. Thegovernment may have certain rights in the invention.

Benefit of provisional application 60/011,545, filed Feb. 13, 1996, ishereby claimed.

BACKGROUND

Legionela pneumophila, the agent of Legionnaires' disease, exists inmany environments such as human alveolar macrophages, fresh waterprotozoa, and biofilms. Cianciotto, et al., Mol. Biol. Med. 6:409-424(1989); Dowling, et al., Microbiol. Rev. 56:32-60 (1992); Fields, TrendsGenet. 4:286-290 (1996); Horwitz, Curr. Top. Microbiol Immunol.181:265-282 (1992). To survive, the bacterium must obtain essentialnutrients from its environment, and the capacity to sense theavailability of a particular nutrient may be important for theregulation of its uptake. Iron is essential for L. pneumophila growthboth extracellularly and intracellularly Byrd and Horwitz, J. Clin.Invest. 83:1457-1465 (1989); Feely, et al., J. Clin. Microbiol.8:320-325 (1978); James, et al., Infect. Immun. 63:4224-4230 (1995);Pine, et al., J. Clin Microbiol. 9:615-626 (1979); Pope, et al., Infect.Immun. 64:629-36 (1996); Reeves, et al., J. Clin Microbiol. 13:688-695(1981)!, serving as a co-factor for an aconitase, a superoxidedismutase, and other enzymes Hoffman, in Legionella: Proceedings of the2nd International Symposium, American Society for Microbiology,Washington, D.C., p. 61-67 (Thornsberry, et al. eds., 1984); Mengaud andHorwitz, J. Bacteriol. 175:5666-5676 (1993); Steinman, Mol. Gen. Genet.232:427-430 (1992)!. L. pneumophila requires 3-13 μM ferric iron forextracellular growth, a level that is relatively high compared to otherbacteria. Hoffman, in Legionella: Proceedings of the 2nd InternationalSymposium, American Society for Microbiology, Washington, D.C., p. 61-67(Thornsberry, et al. eds., 1984); James, et al., Infect. Immun.63:4224-4230 (1995); Johnson, et al., Infect. Immun. 59:2376-2381(1991); Mengaud, et al., J. Bacteriol. 175:5666-5676 (1993); Pine, etal., J. Clin Microbiol. 9:615-626 (1979); Reeves, et al., J. Bacteriol.154:324-329 (1983); Ristroph, et al., J. Clin. Microbiol. 13:115-119(1981). However, iron within the macrophage host is either bound bytransferrin, complexed as ferritin, or sequestered in the labile ironand heme pools. Bridges, in Iron storage and transport, CRC Press, BocaRaton, pp. 297-314 (Ponka, et al. eds., 1990); Crichton andCharloteaux-Wauters, Eur. J. Biochem. 164:485-506 (1987);Muller-Eberhard and Nikkila, Semin. Hematol. 26:86-104 (1989). Thus, theintraphagosomal L. pneumophila likely has evolved strategies forscavenging scarce free-iron or iron bound to cellular chelators.However, the ways in which L. pneumophila obtains iron from itsenvironment remain unknown. High affinity siderophores that are commonlyused for Fe³⁺ assimilation by other bacteria are reported to be absentfrom the legionellae. Liles and Cianciotto, Infect Immun. 64:1873-1875(1996); Reeves, et al., J. Bacteriol. 154:324-329 (1983). Also, thisbacterium does not bind and utilize transferrin or lactoferrin. Bortner,et al., Can. J. Microbiol. 35:1048-1051 (1989); Byrd and Horwitz, J.Clin. Invest. 83:1457-1465 (1989); Byrd and Horwitz, J. Clin Invest.88:1103-1112 (1991); Johnson, et al., Infect. Immun. 59:2376-2381(1991); Quinn and Weisberg, Curr. Microbiol. 17:111-116 (1989). L.pneumophila does, however, express two internal ferric reductases whichlikely process internalized Fe³⁺. Johnson, et al., Infect. Immun.59:2376-2381 (1991); Poch and Johnson, Biometals 6:107-114 (1993).Undoubtedly, the legionellae have multiple iron uptake systems, like somany other pathogens. Guerinot, Annu. Rev. Microbiol. 48:743-72 (1994);Otto, et al., Crit. Rev. Microbiol. 18:217-233 (1992); Payne, TrendsMicrobiol. 1:66-69 (1993); Wooldridge and Williams, FEMS Microbiol Rev.12:325-348 (1993).

In other bacteria, the factors involved in iron uptake, such assiderophores and transferrin receptors, are regulated by the repressorprotein Fur. Bagg and Neilands, Biochemistry 26:5471-5477 (1987); deLorenzo, et al., J. Bacteriol. 169:2624-2630 (1987); Guerinot, Annu.Rev. Microbiol. 48:743-72 (1994); Litwin, Clin. Microbiol. Rev.6:137-149 (1993); Tsolis, et al., J. Bacteriol. 177:4628-37 (1995). Inthe presence of ferrous iron, the Fur dimer binds to promoter regionsand represses the transcription of iron acquisition genes. Bagg andNeilands, Biochemistry 26:5471-5477 (1987); Hantke, Mol. Gen. Genet.182:228-292 (1981). However, when the availability of iron is low, Furdoes not bind and the genes are expressed. In addition to thecontrolling factors involved in iron uptake, Fur represses variety ofother virulence determinants, such as exotoxin A in Pseudomonasaeruginosa and a hemolysin in Vibrio cholerae. Litwin, et al., J.Bacteriol. 174:1897-1903 (1992); Prince, et al., J. Bacteriol.175:2589-2598 (1993).

Recently, the L. pneumophila fur gene was isolated and its predictedamino acid sequence was shown to be 50-60% identical to that of otherbacteria. Hickey and Cianciotto, Gene 143:117-121 (1994). Furthermore,the cloned fur encoded a protein able to repress Escherichia coil fiuexpression and decrease enterobactin production. These data suggestedthat L. pneumophila iron acquisition is regulated by Fur using theconcentration of iron in the environment as a signal for when to repressgene expression.

A number of pathogens, including both intra- and extracellularparasites, have evolved mechanisms for interacting with hemin andheme-containing compounds. Otto, et al., Crit. Rev. Microbiol.,18:217-233 (1992); Wooldridge and Williams, FEMS Microbiol. Rev.12:325-348 (1993). This interaction can serve several functions. First,and perhaps most often, it serves as a means for iron acquisition.Strains of Aeromonas spp., Bacteroides fragilis, Bordetella pertussis,Campylobacter jejuni, Escherichia coli, Haemophilus ducreyi, Haemophilusinfluenzae, Helicobacter pylori, Klebsiella pneumoniae, Neisseriagonorrheae, Neisseria meningitidis, Plesiomonas shigelloides,Porphyromonas gingivalis, Serrati marcescens, Shigella flexneri,Streptococcus pneumoniae, Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, and Yersinia pestis scavenge the iron from hemin andrelated substances. Daskaleros and Payne, Infect. Immun. 55:1393-1398(1987); Daskaleros, et al., Infect. Immun. 59:2706-2711 (1991); Genco,et al., Infect. Immun. 62:2885-2892 (994); Lee, J. Med. Microbiol.34:317-322 (1991); Letoffe, et al., Proc. Natl. Acad. Sci. USA,91:9876-9880 (1994); Massad, et al., J. Gen. Microbiol. 137:237-241(1991); Otto, et al., Crit. Rev. Microbiol. 18:217-233 (1992); Tal, etal., Infect. Immun. 61:5401-5405 (1993); Wooldridge and Williams, FEMSMicrobiol. Rev. 12:325-348 (1993); Worst, et al., Infect. Immun.63:4161-4165 (1995). Second, in H. influenzae and P. gingivalis, hemestructures are absolutely required as sources for porphyrin rings.Carman, et al. Infect. Immun. 58:4016-4019 (1990); Cope, et al., J.Bacteriol. 177:2644-2653 (1995). Third, hemin binding to a bacterialsurface may facilitate infection of eukaryotic cells. For example, in S.flexneri and enteroinvasive E. coli, it enhances invasion of epithelialcells, whereas in Aeromonas salmonicida, hemin interaction with S-layerproteins promotes macrophage association. Daskaleros and Payne, Infect.Immun. 55:1393-1398 (1987); Garduno and Kay, Infect. Immun. 60:4612-4620(1992); Stugard, et al., Infect. Immun. 57:3534-3539 (1989). Fourth,hemin (heme) serves as a cofactor for intracellular cytochromes andenzymes; e.g., the heme-binding FixL of Rhizobium meliloti is anoxygen-sensing membrane kinase. Monson, et al., Proc. Natl. Acad. Sci.USA 89:4280-4284 (1992). Finally, Y. pestis has an extraordinarycapacity to store hemin. Perry, Trends Microbiol. 1:142-147 (1993). Inrecent years, a number of investigators have turned their attentiontoward the molecular and genetic bases of hemin binding and utilization.This complex process generally involves the concerted effort ofsurface/outer membrane receptors and periplasmic/inner membranetransporters and has been characterized as a TonB-dependent uptakeevent. Bramanti and Holt, J. Bacteriol. 175:7413-7420 (1993); Cope, etal., J. Bacteriol. 177:2644-2653 (1995); Elkins, et al., Infect. Immun.63:2194-2200 (1995); Hanson and Hansen, Mol. Microbiol. 5:267-278(1991); Henderson and Payne, J. Bacteriol. 176:3269-3277 (1994); Lewisand Dyer, J. Bacteriol. 177:1299-1306 (1995); Mills and Payne, J.Bacteriol. 177:3004-3009 (1995); Stojiljkovic and Hantke, Mol.Microbiol. 13:719-732 (1994). In S. marcescens, heme acquisition isinitiated by an extracellular heme-binding protein. Letoffe, et al.,Proc. Natl. Acad. Sci. USA 91:9876-9880 (1994).

The relationship between L. pneumophila and hemin has received verylittle attention. Although ferric/ferrous iron clearly plays a criticalrole in extra- and intracellular Legionella growth, Byrd and Horwitz, J.Clin. Invest. 83:1457-1465 (1989); Feeley, et al., J. Clin. Microbiol.8:320-325 (1978); Gebran, et al., Infect. Immun. 62:564-568 (1994);Quinn and Weisberg, Curr. Microbiol. 17:111-116 (1988); Reeves, et al.,J. Clin. Microbiol. 13:688-695 (1981)!, the role of hemin is unclear.Several early studies demonstrated bacterial growth on complex andsemi-defined media which contained hemin or hemoglobin supplements.Feeley, et al., J. Clin. Microbiol. 8:320-325 (1978); Pine, et al., J.Clin. Microbiol. 9:615-626 (1979). However, since the heme compoundswere easily replaced with ferric salts, it was assumed that they werenot required for Legionella growth. Feeley, et al., J. Clin. Microbiol.8:320-325 (1978). This idea was later confirmed by the development ofdefined media which completely lacked hemin but supported effective L.pneumophila replication. Reeves, et al., J. Clin. Microbiol. 13:688-695(1981); Ristroph, et al., J. Clin. Microbiol. 13:115-119 (1981); Warrenand Miller, J. Clin. Microbiol. 10:50-55 (1979). Nevertheless, hemin andhemoglobin did enhance L. pneumophila growth on several types of complexmedia. Current protocols in molecular biology (Ausubel, et al., eds.,1987); Johnson, et al., J. Clin. Microbiol. 15:342-344 (1982). In onestudy, the growth of seven L. pneumophila strains, representing sixserogroups, was stimulated by ≧100-fold by the addition of hemin to ayeast extract phosphate (YP) medium. Johnson, et al., J. Clin.Microbiol. 15:342-344 (1982). Taken together, these data suggest thathemin can serve as an accessory iron source.

SUMMARY OF THE INVENTION

The invention comprises the frgA genes of Legionella pneumophila. FrgAis conserved in, and is specific to, L. pneumophila. Thus, the inventionalso comprises methods and materials for detecting L. pneumophila, themethods and materials being targeted to a frgA gene, an mRNA encoded bya frgA gene, or a frgA protein. The materials include frgA-specificprobes and polymerase chain reaction (PCR) primers and antibodies thatbind specifically to frgA proteins. The materials may be supplied in kitform.

The invention also comprises the hbp gene of L. pneumophila. The gene isfound almost exclusively in L. pneumophila, and the invention comprisesother methods and materials for detecting L. pneumophila, the methodsand materials being targeted to an hbp gene, an mRNA encoded by a hbpgene, or an hbp protein. These materials include hbp-specific probes andPCR primers and antibodies that bind specifically to hbp proteins. Thematerials may also be supplied in kit form.

The frgA and hbp genes are exceptional for their unique, limiteddistribution among legionellae. All previously identified L. pneumophilagenes, including flaA, fur, htp, lly, mip, omps, and pplA (pal), havehad homologs in all or virtually all other Legionella species. Bender,et al., Infect. Immun. 59:3333-6 (1991); Cianciotto, et al., InfectImmun. 58:2912-2918 (1990); Heuner, et al., Infect. Immun. 63:2499-507(1995); Hickey and Cianciotto, Gene 143:117-121 (1994); Hoffman, et al.,Infect. Immun. 57:1731-1739 (1989); Hoffman, et al., J. Bacteriol.174:914-920 (1992); Ott, et al., Microb. Pathog. 11:357-65 (1991).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Map of plasmid pEH40, the plasmid used for miniTn10'lacZmutagenesis of L. pneumophila. The ca. 10-kb pEH40 contains: i) aminiTn10 (see IS10 elements) which carries a promoterless lacZ ('lacZ)and a kanamycin-resistance gene (Km-r); ii) a doubly mutated Tn10transposase gene (atsIats2) which promotes increased variability of thetransposon insertions compared to the wild-type transposase Kleckner, etal., in Methods in Enzymology, 204, p. 139-180 (Miller ed. 1991)!; iii)the counterselectable levansucrase gene of Bacillus subtilis (sacB)Cianciotto, et al., FEMS Microbiol. Letts. 56:203-208 (1988)!; and iv) aColE1 origin of replication (oriV). Key restriction sites are indicated.

FIG. 2. Southern blots. Genomic DNAs of strains NU229 through NU236,created by miniTn10'lacZ insertions in L. pneumophila, were digestedwith HindIII, electrophoresed, and hybridized with pEH40. Lanes: A,NU229; B, NU230; C, NU231; D, NU232; E, NU233; F, NU234; G, NU235; andH, NU236. Molecular weight markers, in kb, are indicated on the left.Since HindIII cuts asymmetrically within mini-Tn10'lacZ (see FIG. 1),one hybridizing fragment appears more intense than the other within eachlane. The intensity of the bands varied between the lanes because notall lanes contained identical amounts of DNA. Although the hybridizingbands in lane C do not appear, in this gel, to comigrate with those inlanes A and B, additional blots using HindIII and EcoRI digested DNAsillustrate that the corresponding strains do have identical insertions.Note that lane F has a faint second band at approximately 2.1-kb, whilelane H has one at approximately 12-kb.

FIGS. 3A-B. Graphs of β-galactosidase production in L. pneumophila lacZfusion strains. Strains were grown for 48 hours onbuffered-charcoal-yeast-extract (BCYE) agar with the standard ironsupplement (solid bars) or on BCYE agar with a ferric iron chelator(hatched bars). Whereas the experiment depicted in FIG. 3A utilized 50μM iron chelator deferoxamine mesylate (DFX), the one in FIG. 3B used 52μM iron chelator ethylenediamine di(o-hydroxyphenylacetic acid) (EDDA).FIGS. 3A and 3B are representative of two and three separate trials,respectively. In every trial, all samples were tested in triplicate.

FIGS. 4A-D. Graphs of OD₆₆₀ versus time. Wild-type strain 130b () andmutant strain NU229 (∘) were grown in chemically defined medium (CDM)with increasing iron concentrations. FIGS. 4A-D depict growth ofcultures containing 0, 4, 8, and 16 μM of added iron, respectively.Bacteria used to inoculate the cultures had been grown overnight inbuffered-yeast-extract (BYE) broth without the iron supplement andwashed once with CDM (0 μM added iron). Growth was estimated bymeasuring the OD₆₆₀ of the culture over a 7-day period. FIGS. 4A-D arerepresentative of three separate trials, and each timepoint is theaverage of two separate cultures. Growth in CDM containing a 20 μM ironsupplement was not different from growth with medium having 60 μM iron(data not shown).

FIGS. 5A-B. Graphs of colony forming units (CFU) per monolayer versustime after inoculation of U937 cells with L. pneumophila mutants. InFIG. 5A, the monolayers (n=4) were inoculated with 10⁶ CFU of eitherstrain 130b (), strain NU229 (∘), or strain NU229 R (Δ). After variousincubation periods, the numbers of bacteria were determined. In FIG. 5B,the U937 cells were inoculated with 10⁶ CFU of either 130b(pSU2719) (),NU229 (pSU2719) (∘), or NU229 R(pEH75) (Δ). All time-points representthe mean CFU recovered, and vertical bars indicate standard deviation.The differences in the recoveries of these strains were significant atall timepoints without overlapping error bars (P<0.01, Student'st-test).

FIG. 6. Restriction map of the L. pneumophila DNA containing theinterrupted frgA gene. The cloned 12-kb region containing L. pneumophilaDNA and the transposon insert is depicted on the top line. Thetransposon consists of the thin arrows, which represent the 'lacZ andKm^(r) genes, and the black circles indicating the location of the IS10elements. The thick line flanking the transposon represent theinterrupted frgA gene. Probe B is frgA specific, containing only frgADNA in addition to the IS10 element. Restriction enzymes' abbreviationsare BamHI (B), EcoRI (E), HindIII (H), KpnI (K), PstI (P), XbaI (Xb),and XhoI (X).

FIG. 7. DNA and predicted amino acid sequences of L. pneumophila frgASEQ ID NO:4!. The potential ribosome binding site is boxed. The -35 and-10 regions are in bold and labeled. Two overlapping ironboxes areunderlined or double-underlined and have 4 or 5 mismatches,respectively. The target sequence that was duplicated as a result ofminiTn10'lacZ insertion is indicated with a black box. Recognition sitesfor EcoRI and XhoI are labeled and italicized. The L. pneumophila frgAsequence has been deposited in the GenBank database at the NationalCenter for Biotechnology Information (NCBI) under accession numberU76559.

FIG. 8. comparison of sequence of FrgA of L. pneumophila with sequencesof IucA and IucC of E. coli SEQ ID NOS:6-44!. The E. coli sequences werereported previously. Martinez, et al., J. Mol. Biol. 238:288-293 (1994).Identical amino acids are indicated with a vertical line, while similaramino acids, as defined by PCGene, are indicated with a black circle.The three boxed and three shaded regions show the homologous sequencesindicated in the BLASTX results from the NCBI. In these regions only,additional conserved amino acids, as defined by the BLAST program, areindicated with a small, lowered dot. Note that these regions overlap theareas that are most similar between IucA and IucC. Martinez, et al., J.Mol. Biol. 238:288-293 (1994).

FIG. 9. Southern blots showing hybridization of DNAs from Legionellaspecies with an frgA probe. DNAs were digested and electrophoresedthrough 0.8% agarose. A Southern blot was then hybridized with probe B(see FIG. 6) under high-stringency (left half of figure) andlow-stringency (right half of figure) conditions.

FIG. 10. Graph of % hemin bound by wild-type L. pneumophila versus theamount of hemin. A total of 1.0×10⁸ CFU of strain 130b obtained fromYP-minus-Fe-plus-hemin agar (∘) and 1.6×10⁸ CFU harvested from BCYE agar(574 ) were assayed for their ability to remove hemin from the solution.Each datum point represents the mean percent hemin bound for threereplicate cultures, and the vertical bars denote the standarddeviations. The differences in binding between theYP-minus-Fe-plus-hemin and BCYE cultures were significant at each heminconcentration (P=<0.001 for all concentrations except 5 μg. for whichP=<0.01 Student's t test!.

FIGS. 11A-B. Graphs of hemin bound by recombinant E. coli and mutant L.pneumophila versus amount of hemin added. FIG. 11A: E. coliHB101(pBR322) (open box) and HB101(pEH1) (shaded box) were grown tostationary phase in M9CA salts broth, and then about 10⁹ CFU of eachwere assayed for their ability to remove hemin from solution. Each datumpoint represents the mean binding for three replicate cultures, and thevertical bars denote the standard deviations. The differences in bindingbetween the two strains were significant at both hemin concentrations(P=<0.001 Student's t test!). HB101(pEH2) and HB101(pBOC3) exhibitedhemin-binding capacities that were comparable to that of HB101(pEH1)(data not shown). FIG. 11B. L. pneumophila 130b (open box) and NU226(shaded box) were grown on YP-minus-Fe-plus-hemin agar plates, and thenabout 10⁸ CFU were assayed for hemin binding. Significant differences inbinding were evident at all hemin concentrations, including the 20-μg/mllevel not depicted here (P=<0.001 Student's t test!).

FIG. 12. Identification of the L. pneumophila hbp ORF; restriction mapsand phenotypes for pEH1 and its subclones. All recombinant plasmidsrepresent Legionella DNA cloned into pBR322+ and -, the HB101transformant was or was not pigmented on agar media containing hemin orCongo red, respectively. Restriction enzyme recognition sites areindicated as follows: A, AflII; Ac, AccI; B, BamHI; E, EcoRI; H, HincII;N, NdeI; P, PstI; and S, SacI.

FIGS. 13A-B. Mutagenesis of the hbp gene of L. pneumophila. FIG. 13A:Diagram of the mutagenesis procedure. The top half depicts apBOC22-containing L. pneumophila transformant. The dashed line indicatesa double-crossover event between the plasmid and the chromosome. Thepredicted result of this recombination, with the mutated hbp replacingthe wild-type gene, is shown at the bottom. The locations of BamHI,HincII, NdeI, and SacI recognition sites are indicated. (H), the HincIIsite that was lost upon insertion of Km^(r) into hpb. FIG. 13B:Demonstration of allelic exchange by Southern hybridization analysis.Genomic DNAs were digested with either HincII (lanes a and b) or NdeI(lanes c and d) and were probed with ³² P-labeled pBOC22. Strain 130bappears in lanes a and c, and strain NU226 is represented in lanes b andd. The migrations and sizes (in kilobases) of molecular markers areindicated.

FIG. 14. Graph of CFU per monolayer versus time post-inoculation forintracellular infection of U937 cells by strains of L. pneumophila. U937cell monolayers (n=4) were inoculated with 10⁵ CFU of either strain130(b) () or strain NU226 (∘), and after various incubation periods,the numbers of viable intracellular bacteria were determined. Since 2 hwere allowed for bacterial attachment and entry, the first sample thatwas collected is presented as a 2-h datum point. Each point representsthe mean CFU recovered, and the vertical bars indicate the standarddeviations.

FIGS. 15A-B. Southern blots showing hybridization of DNAs fromLegionella species with an hbp probe. DNAs were digested with EcoRI andelectrophoresed through 0.8% agarose. A Southern blot was thenhybridized with the hbp-containing Ndel-EcoRV fragment of pEH12 underhigh- (FIG. 15A) and low- (FIG. 15B) stringency conditions. FIG. 15A:Lanes: a and b, L. pneumophila serogroup 1 strains 130b and Philadelphia1, respectively; c and d, L. pneumophila serogroups 8 and 13,respectively. FIG. 15B: Lanes: a, L. pneumophila 130b; b, L. micdadei;c, L. erthyra; d, L. feeleii; e, L. hackeliae; f, L. longbeachae; g, L.moravica; h, L. santicnisis; and i, L. spiritensis. The migrations andsizes (in kilobases) of molecular weight markers are indicated.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

The invention provides a frgA gene of Legionella pneumophila. Inparticular, Example 1 describes the identification, isolation andcharacterization of the frgA gene of L. pneumophila strain 130b.Southern hybridization analysis using this gene showed that frgA genesare conserved in, and specific to, L. pneumophila (see Example 1). Thesequence of the coding region of the frgA gene of strain 130b andportions of the flanking regions is given in FIG. 7 SEQ ID NO:4!.

As used herein a "frgA gene" is an L. pneumophila gene that hybridizesto the frgA gene of strain 130b, or a fragment thereof at least 18basepairs (bp) in length, under conditions of high stringency."High-stringency conditions" are defined herein to be those conditionsgiving hybridization only if there are about 10% or less bp mismatches.

FrgA genes other than the frgA gene of strain 130b can be identified andisolated using methods well known in the art. For instance, probes basedon the sequence of the frgA gene of strain 130b can be used to screen L.pneumophila genomic libraries for other frgA genes.

Since, as noted above, frgA genes are conserved in, and specific for, L.pneumophila strains, these genes, and the mRNAs and proteins encoded bythem, are excellent targets for the detection or quantitation of L.pneumophila strains. For instance, L. pneumophila may be detected orquantitated by polymerase chain reaction (PCR) using primers that willamplify at least a portion of a frgA gene, by the use of DNA or RNAprobes that will hybridize to a frgA gene or mRNA, or by an immunoassayusing antibodies that bind specifically to a frgA protein.

To perform the PCR assay, DNA is obtained from a sample suspected ofcontaining L. pneumophila. Also, mRNA may be obtained from such a sampleand used to prepare cDNA which can be used as the template in the PCR.Methods of extracting total cellular DNA and RNA from cells and methodsof preparing cDNAs are well known. However, PCR does not require highlypurified DNA, and DNA released by boiling of cells can be used directlywithout any purification.

The PCR primers are nucleic acid molecules having sequences selected sothat the molecules hybridize to one of the strands of a frgA gene. Ofcourse, at least two primers must be used (one hybridizing to each ofthe strands of the frgA gene), but more than one pair of primers can beused if it is desired to amplify more than one portion of the frgA gene.The primers should be at least 18-20 bp in length with a G+C contentgreater than 40%. The specificity of the primers should be confirmed bySouthern blotting.

Next, the DNA is amplified by PCR. PCR methods, equipment, and reagentsare well known and are available commercially.

Finally, the amplified DNA is detected or quantified. This can beaccomplished in a number of ways as is known in the art. For instance,the reaction mixture can be electrophoresed on agarose gels, and thepresence or absence of amplified DNA of the expected size(s) can bedetermined by staining the gels. Alternatively, a labeled nucleic acidmolecule which hybridizes to the amplified DNA (a probe) can be used toallow for detection or quantitation of the amplified DNA. As anotheralternative, the primers can be labeled, or the nucleotides used duringthe PCR can be labeled, and the labels incorporated into the amplifiedDNA can be detected or quantified.

Legionela pneumophila can also be detected or quantitated by obtainingDNA or RNA from a sample suspected of containing L. pneumophila. Thiscan be accomplished in the same manner as described above for PCRanalysis. The DNA or RNA is contacted with a probe which is a nucleicacid molecule having a sequence selected so that the molecule hybridizesto a frgA gene or to mRNA encoded by a frgA gene. The probe is allowedto hybridize with the DNA or RNA obtained from the sample. To detect orquantitate the frgA gene or MRNA, the probe is labeled. The probe shouldbe as large as possible while retaining specificity.

The invention further comprises the nucleic acid molecules used asprobes and primers in these techniques. These nucleic acids moleculeshave sequences selected so that the molecules hybridize to a frgA geneor an MRNA encoded by a frgA gene. For instance, a suitable probe orprimer may have the sequence of the frgA gene given in FIG. 7 SEQ IDNO:4! or a fragment thereof. These nucleic acid molecules could alsoinclude antisense RNA molecules and ribozymes. Methods of making nucleicacid molecules are, of course, well known in the art.

The probes or primers may be labeled to allow for detection orquantitation of L. pneumophila. Suitable labels and methods of attachingor incorporating them into nucleic acid molecules are well known.Suitable labels include radioactive labels (e.g., ³² P),chemiluminescent labels, fluorescent labels (e.g., fluorescein,rhodamine), particulate labels (e.g., gold colloids), colorimetriclabels (e.g., dyes), enzymes, and biotin.

As noted above, labeled nucleotides can also be used during PCR togenerate an amplified DNA which is labeled. The nucleotides arepreferably labeled with radioactive labels (e.g., ³² P) by methods wellknown in the art.

The invention also provides a kit containing reagents useful fordetecting or quantifying L. pneumophila. The kit comprises at least onecontainer holding at least one nucleic acid molecule of the invention (aprobe or primer). For PCR assays, the kit will comprise at least twoprimers, which may be in the same container or in separate containers.The probes or primers may be labeled. The kit may contain other reagentsand equipment useful in performing the assay, including PCR reagents(e.g., polymerase, labeled or unlabeled nucleotides), reagents forextraction of DNA or RNA, buffers, salt solutions, containers, gels andmembranes, etc.

L. pneumophila may also be detected or quantitated in an immunoassayusing antibodies that bind specifically to a frgA protein. FrgA proteinsor peptides can be used to produce antibodies useful in the immunoassayby methods well known in the art. A frgA gene or fragment thereof may beused to produce a frgA protein or peptide. Peptides can also be preparedby solid phase synthetic methods. Single chain or other engineeredantibodies or fragments of antibodies containing an antibody combiningsite can be also used.

To perform the immunoassay, a sample suspected of containing L.pneumophila is contacted with the antibody under conditions so that theantibody can bind to a frgA protein, if present. Since frgA proteins arebelieved not to be found on the surface of L. pneumophila, the cellswill likely have to be lysed to release the protein. Standardimmunoassay formats can be used. The antibody bound to the frgA proteinmay be labeled to allow for detection or quantitation of the frgAprotein, or a labeled moiety that binds to the antibody (e.g., asecondary antibody directed to the first antibody or protein A) may beused. Suitable labels and methods of attaching them to antibodies andother proteins and compounds are well known. Suitable labels includeradioactive labels (e.g., ¹²⁵ I), fluorescent labels (e.g., fluorescein,rhodamine), chemiluminescent labels, particulate labels (e.g., goldcolloids), colorimetric labels (e.g., dyes), enzymes, and biotin.

Reagents useful for detecting or quantifying L. pneumophila byimmunoassay may also be supplied as a kit. The kit will comprise atleast one container holding an antibody that binds specifically to afrgA protein. The kit may contain other reagents and equipment useful inperforming immunoassays, including buffers, containers (e.g., testtubes, culture plates), substrates for enzyme labels, labeledstreptavidin or avidin to bind to a biotin label, etc.

The invention further provides an hbp gene of L. pneumophila. Inparticular, Example 2 describes the identification, isolation andcharacterization of the hbp gene of L. pneumophila strain 130b. Southernhybridization analysis using this gene showed that hbp genes are foundalmost exclusively in L. pneumophila strains. Under low stringencyconditions, weak hybridization of an hbp probe to genes in twoLegionella species was observed. The sequence of the coding region ofthe hbp gene of strain 130b and portions of the flanking regions isgiven in Chart A SEQ ID NO:45!.

As used herein an "hbp gene" is an L. pneumophila gene that hybridizesto the hbp gene of strain 130b, or a fragment thereof at least 18 bp inlength, under conditions of high stringency.

Hbp genes other than the hbp gene of strain 130b can be identified andisolated using methods well known in the art. For instance, probes basedon the sequence of the hbp gene of strain 130b can be used to screen L.pneumophila genomic libraries for other hbp genes.

Since, as noted above, hbp genes are found almost exclusively in L.pneumophila strains, hbp genes, and the mRNAs and proteins encoded bythem, are good targets for the detection or quantitation of L.pneumophila strains. For instance, L. pneumophila may be detected orquantitated by polymerase chain reaction (PCR) using primers that willamplify at least a portion of an hbp gene, by the use of DNA or RNAprobes that will hybridize to an hbp gene or mRNA, or by an immunoassayusing antibodies that bind specifically to a hbp protein. In particular,if high-stringency conditions are used for hybridizing probes to an hbpgene, mRNA or PCR-amplified DNA, the assays will differentiate L.pneumophila from other Legionella species (see Example 2). Also, the useof hbp-specific probes should also enhance the specificity of PCR assaysas compared to current assays using primers directed to genes found inall Legionella species (see Example 2, particularly the finalparagraph). These assays are performed as described above for the frgAgene, mRNA and protein. Also, reagents for performing these assays arethe same as described above, except that they are targeted to an hbpgene, an mRNA encoded by an hbp gene, or an hbp protein. The reagentsmay be supplied in kit form as described above.

Samples that may be used in the methods of the invention include watersamples and clinical samples. Water samples may be taken from anysource, including plumbing fixtures, evaporative condensers, coolingtowers and supplies of potable water (e.g., lakes and rivers). Clinicalsamples include sputum, throat swabs, blood, urine, cerebrospinal fluid,skin, biopsies, saliva, synovial fluid, bronchial lavages, or othertissue or fluid samples from humans or other animals.

Many of the procedures described above utilize hybridization of nucleicacids. Hybridization conditions and guidelines for selecting high- andlow-stringency conditions are well known in the art. See, e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual (2nd ed. 1989). Suitablehybridization conditions can be readily determined and optimized usingonly ordinary skill in the art.

The above-described assays targeted to a frgA gene, an MRNA encoded by afrgA gene, or a frgA protein, or to an hbp gene, an MRNA encoded by anhbp gene, or an hbp protein may be used alone or in combination witheach other or with other tests to detect or quantitate L. pneumophila.For instance, a PCR assay could utilize primers directed to a genecommon to all Legionella in combination with primers directed to thefrgA gene which is specific to L. pneumophila.

EXAMPLES Example 1 Cloning and Mutation of L. pneumophila frg Genes

Using a new random mutagenesis protocol, the L. pneumophila loci thatare regulated by iron were identified. In particular, wild-type L.pneumophila strain Wadsworth 130b was randomly mutated with aminiTn10'lacZ transposon. The resulting gene fusions were tested foriron-regulation by assessing β-galactosidase production in the presenceand absence of iron chelators. Of the initial six mutants withiron-repressed lacZ fusions, two strains, NU229 and NU232, possessedfusions that were stably iron-regulated.

To assay for Fur regulation, the levels of β-galactosidase were measuredin strains no longer producing Fur. As in a number of pathogenicbacteria, L. pneumophila fur could not be insertionally inactivated, butspontaneous Fur⁻ derivatives were generated by selecting for manganeseresistance. Strain NU229 contained a Fur-repressed fusion based onderepression of lacZ expression in its manganese-resistant derivative.Extracellular growth of NU229 in bacteriological media was similar tothat of wild-type strain 130b.

To assess the role of an iron- and Fur-regulated (frgA) gene inintracellular infection, the ability of NU229 to grow within U937 cellmonolayers was tested. Quantitative infection assays demonstrated thatNU229 was impaired as much as 80-fold in intracellular growth.Reconstruction of the mutant by allelic exchange proved that theinfectivity defect in NU229 was due to the inactivation of frgA and nota second-site mutation. Subsequently, complementation of the interruptedgene by an intact plasmid-encoded gene demonstrated that the infectivitydefect was due to the loss of frgA and not a polar effect.

Nucleotide sequence analysis revealed that the 63 kD FrgA has homologywith the aerobactin synthetases IucA and IucC of Escherichia coli,raising the possibility that L. pneumophila encodes a siderophore whichis required for optimal intracellular replication.

Southern hybridization analysis determined that frgA is specific to theL. pneumophila species and is not found in other Legionella species.

Portions of this work were previously presented. Hickey and Cianciotto,in Abstracts of the 95th General Meeting of the American Society forMicrobiology, Abstract B-375, page 230 (1995).

A. Materials and Methods

1. Bacterial Strains, Media, and Chemicals. The L. pneumophila frg geneswere cloned from and inactivated within serogroup 1 strain 130b. OtherL. pneumophila strains tested for the presence of frgA includedserogroup 2 strain ATCC 33154, serogroup 3 strain ATCC 33155, serogroup4 strain ATCC 33156, serogroup 7 strain ATCC 33823, serogroup 8 strainATCC 35096, serogroup 13 strain B2A3105, serogroup 14 strain 1169-MN-H,as well as representatives of serogroups 9-12, that were obtained fromthe Michigan Department of Public Health. Cianciotto, et al., InfectImmun. 58:2912-2918 (1990). Other Legionella species examined include L.birminghamensis 1407-AL-H, L. dumoffii ATCC 33279, L. erytha SE-32A, L.gormanii ATCC 33297, L. feeleii WO-44C, L. hackeliae Lansing 2, L.israelensis Bercovier 4, L. jamestowniensis JA-26, L. longbeachae ATCC33462, L. micdadei Rivera, L. oakridgensis OR-10, L. parisiensis PF-209,L. sainthelensi Mt. St. Helen's 4, L. santicrusis SC-63, L. spiritensisMSH-9, and L. tucsoniensis 1087-AZ-H. Cianciotto, et al., Infect Immun.58:2912-2918 (1990).

All Legionella strains were grown at 37° C. onbuffered-charcoal-yeast-extract (BCYE) agar for 48-72 hours or withinbuffered-yeast-extract (BYE) broth. Cianciotto, et al., Infect Immun.58:2912-2918 (1990). When appropriate, 25 μg/ml kanamycin, 3 μg/mlchloramphenicol, 5%(w/v) sucrose, or 0.5 μg/ml streptonigrin was addedto the medium. To select and maintain L. pneumophila Fur mutants,0.4-0.6 mM manganese chloride was added to BCYE instead of the ironsupplement. To generate low-iron BCYE, 50-60 μM of deferoxamine mesylate(DFX) or 50-80 μM of ethylenediamine di(o-hydoxyphenylacetic acid)(EDDA) was added in place of the standard 355 μM ferric pyrophosphatesupplement. These levels of Fe³⁺ chelators did not significantly inhibitbacterial growth. Chemically defined medium (CDM) and acid washedglassware were used for monitoring L. pneumophila growth kinetics inliquid. Reeves, et al., J. Bacteriol. 154:324-329 (1983). The indicatedconcentrations of iron within CDM were achieved by adding ferricpyrophosphate supplements. E. coli strain HB101 served as a host forrecombinant plasmids and was grown on Luria-Bertani agar containing 50μg/ml kanamycin, 50 μg/ml ampicillin, or 30 μg/ml chloramphenicol.Current protocols in molecular biology (Ausubel, et al., eds. 1987).Unless stated otherwise, all chemicals were obtained from Sigma ChemicalCo., St. Louis, Mo.

2. Random Mutagenesis of L. pneumophila with MiniTn10'lacZ

To identify L. pneumophila genes regulated by iron, promoterless lacZ('lacZ) genes were randomly inserted into the 130b chromosome. PlasmidpEH40 (FIG. 1), which was used to introduce miniTn10'lacZ into L.pneumophila was constructed in two steps. First, a 'lacZ cassette,isolated from BamHI-digested pNK2804, was ligated to the BglII site inthe miniTn10 of pGI6145. Kleckner, et al., in Methods in Enzymology,204, 139-180 (Miller, ed. 1991). Then, the resulting plasmid wasdigested with PstI and ligated with the sacB-containing PstI fragmentfrom pUCD800+oriT. Cianciotto, et al., FEMS Microbiol. Letts. 56:203-208(1988).

To isolate miniTn10'lacZ-containing legionellae, pEH40 waselectroporated into strain 130b, and transformants were selected on BCYEagar containing kanamycin and sucrose. Cianciotto, et al., Proc. Natl.Acad. Sci. USA 89:5188-5191 (1992); Pope, et al., FEMS Microbiol. Letts.124:107-112 (1994). The frequency of kanamycin-resistant (Km^(r)),sucrose-resistant colonies was 6.4×10⁻⁶, suggesting that thetransposition frequency of miniTn10'lacZ is comparable to that ofminiTn10 without 'lacZ. Pope, et al., FEMS Microbiol. Letts. 124:107-112(1994). To identify those mutants that produced β-galactosidase, thebase plates were topped with 0.7% agar containing 0.6 mg/ml5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside puriss (XGAL).Miller, Experiments in Molecular Genetics (1972). Approximately 46% ofKm^(r), sucrose-resistant colonies exhibited blue pigmentation. Toquantitate the degree of lacZ expression in L. pneumophila,β-galactosidase activity was measured as described in Miller,Experiments in Molecular Genetics (1972). In preparation for the assay,strains were grown for 48-72 hours on BCYE agar and then suspended insterile water to an optical density at 660 nm (OD₆₆₀) of 0.4-0.7.

3. Directed Mutagenesis and Trans-complementation of L. pneumophila.

To mutate specific genes in the L. pneumophila chromosome, the clonedgenes, inactivated with Km^(r) inserts, were ligated into thecounterselectable pEA75, and allelic exchange was performed as describedpreviously. Cianciotto, et al., FEMS Microbiol. Letts. 56:203-208(1988); O'Connell, et al., Infect. Immun. 63:2840-2845 (1995). Forattempts at interrupting L. pneumophila fur, the cloned gene wasobtained from plasmid pEH22a. Hickey and Cianciotto, Gene 143:117-121(1994). To complement a chromosomal mutation, the relevant L.pneumophila gene was cloned into the pACYC184 derivative pSU2719,transferred into the mutant by electroporation, and maintained withchloramphenicol selection. Cianciotto, et al., FEMS Microbiol. Letts.56:203-208 (1988); Martinez, et al., Gene 68:159-162 (1988).

4. Isolation of L. pneumophila Genomic DNA.

Genomic DNA was isolated from L. pneumophila strains using a protocoladapted from Neumann, et al., Trends Genet. 8:332-333 (1992). Thebacteria were swabbed from a fresh agar plate and resuspended in 1 ml ofsterile water. After a 2 minute centrifugation (16,000×g), the bacterialpellet was suspended in 0.5 ml SET (75 mM NaCl, 25 mM EDTA, 20 mM TrispH 7.5) containing 1 mg/ml lysozyme and 0.1 mg/ml RNase A. The mixturewas incubated for 30 minutes at 37° C. before adding 0.1 volumes of 10%sodium dodecyl sulfate (SDS) and 0.05 volumes of 20 mg/ml Proteinase K.Following incubation at 55° C. for 2 hours, one third volume ofNaCl-saturated water was added to the tube. Next, one volume of 0.1MTris (pH 8)-equilibrated phenol was added, and the mixture incubated atroom temperature for 30 minutes using a rotator. The phases wereseparated by a 5-minute centrifugation (16,000×g), and the aqueous phasewas removed to a new tube. Two additional extractions were performedusing 1 volume of phenol/chloroform/isoamyl alcohol (25:24:1) and then 1volume of chloroform/isoamyl alcohol (24:1). Finally, after removing theaqueous phase to a new tube, the DNA was precipitated by adding 1 volumeof isopropanol.

5. DNA Hybridizations.

For Southern blot hybridizations, genomic DNAs from L. pneumophilastrains were digested with restriction enzymes according to themanufacturer's protocols (New England Biolabs, Beverly, Mass.). DNAswere then electrophoresed through 0.8% agarose and transferred to anitrocellulose filter. Current protocols in molecular biology (Ausubelet al., eds., 1987). Digoxigenin-labeling of the indicated probes andhigh-stringency hybridizations (i.e., ca. 10% basepair mismatch) wereperformed using the Genius System v2.0 (Boehringer Mannheim,Indianapolis, Ind.). Two modifications were made to the Genius Systemprotocols to achieve low stringency conditions (i.e., ca. 30% basepairmismatch). First, the hybridization and wash temperatures were droppedto 49° C. and, second, the sodium chloride-sodium citrate (SSC)concentration in the wash buffer was raised to 5× (750 mM NaCl, 75 mMsodium citrate; pH 7.0).

For colony blot hybridizations, an L. pneumophila 130 genomic library,consisting of 4-6 kb Sau3A1 fragments cloned into pBR322 Currentprotocols in molecular biology (Ausubel et al., eds., 1987)!, was platedon LB-ampicillin agar for isolation of colonies. The colonies weretransferred to nitrocellulose and lysed by incubating the membranes onWhatman paper saturated with 10% SDS, followed by subsequent incubationson denaturing solution (0.5N NaOH, 1.5M NaCl), neutralizing solution (1MTris-HCl, pH 8.0, 1.5M NaCl), and 2× SSC. All incubations were performedat room temperature for five minutes. Digoxigenin-labeled probes andhigh-stringency hybridizations were performed as above.

6. Western Blot.

Whole cell lysates were harvested and reacted with antiserum asdescribed previously. Cianciotto, et al., Infect Immun. 58:2912-2918(1990). Antiserum against E. coli Fur was received from Michael Vasiland used at a 1/100 dilution. Prince, et al., J. Bacteriol.175:2589-2598 (1993). Horseradish peroxidase-conjugated secondaryantibody was used at a 1/1000 dilution (Gibco BRL, Gaithersburg, Md.).

7. Intracellular Infection of U937 Cells.

U937 is a human cell line that differentiates into macrophage-like cellsafter treatment with phorbol esters. Pearlman, et al., Microb. Pathog.5:87-95 (1988). U937 cell monolayers were prepared and infected aspreviously described (Pearlman, et al., Microb. Pathog. 5:87-95 (1988)).After inoculation, the monolayers were incubated for 2 hours to permitbacterial uptake and then washed to remove unattached bacteria. Theinfected monolayers were incubated at 37° C. in RPMI medium supplementedwith 10% fetal bovine serum (Gibco BRL). To assess the relativeinfectivity of L. pneumophila strains, 50% infective doses (ID₅₀) weredetermined after a 3-day incubation. Cianciotto, et al., Infect. Immun.,57:1255-1262 (1989); O'C.onnell, et al., Infect. Immun., 63:2840-2845(1995). To quantitate intracellular bacteria, replicate (n=4) monolayerswere inoculated with approximately 10⁶ colony forming units (CFU),incubated for 24-72 hours, and then lysed. O'C.onnell, et al., Infect.Immun. 63:2840-2845 (1995). Tenfold serial dilutions of the lysates wereplated on BCYE agar, containing chloramphenicol where appropriate, andthe resulting CFU were used to calculate the corresponding numbers ofbacteria per monolayer.

8. DNA Sequence Analysis.

Cloned L. pneumophila DNA was sequenced from double-stranded plasmids bythe dideoxy chain termination method using ³⁵ S!dATP (Amersham LifeScience, Arlington Heights, Ill.) and Sequenase (United StatesBiochemical, Cleveland, Ohio). Current protocols in molecular biology(Ausubel et al., eds. 1987). Initially, M13-based primers (5'-CCCAGTCACGACGTTGTAAA ACG SEQ ID NO: 1! and 5'-AGCGGATAAC AATTTCACAC AG SEQ ID NO:2!) and an IS10-based primer (5'-CCTTAACTTA ATGATTTTTA C SEQ ID NO: 3!)were used to sequence DNAs cloned into pSU2719, which contains lacZ andthe multicloning site from pUC19. Andrews, et al., J. Bacteriol.171:3940-3947 (1989). Twenty custom 18- to 20-mer oligonucleotideprimers were used in subsequent reactions. The primers were prepared bythe Northwestern University Biotechnology Center using the AppliedBiosystems DNA synthesizer (Perkin-Elmer, Foster City, Calif.).Sequencing reactions were performed according to the manufacturer'sprotocols and the olignonucleotides were electrophoresed in Long-Ranger(AT Biochem, Malvern, Pa.) acrylamide gels. Both strands of DNA weresequenced and then analyzed using PCGene (Intelligenetics, MountainView, Calif.). Nucleotide and predicted amino acid sequences, as well asinitial homology studies, were obtained from GenBank at the NationalCenter for Biotechnology Information (NCBI), National Library ofMedicine, NIH. Sequence homologies on the complete predicted amino acidsequences were performed using PCGene. The frgA sequence is deposited inthe GenBank database, NCBI, under accession number U76559.

B. Results

1. Identification of Iron-repressed Genes in L. pneumophila.

To identify iron-repressed genes of L. pneumophila, strain 130b wasmutated with miniTn10'lacZ and screened for strains that had the highestβ-galactosidase expression under low-iron growth conditions.Approximately 700 colonies, which appeared white to light blue onstandard BYCE agar overlaid with XGAL, were replica plated onto BCYEagar containing 50 μM DFX. The chelator DFX has its greatest affinity(i.e., 10³¹) for Fe³⁺, while its next highest affinity (i.e., 10¹⁴) isfor Cu²⁺. Keberle, Ann N.Y. Acad. Sci. USA 119:758-768 (1964). Eightmutants, designated as strains NU229 through NU236, consistentlyexpressed higher levels of β-galactosidase activity on the low-ironmedium, suggesting that they contained iron-repressed lacZ fusions.Southern blot analyses were performed to determine the number andlocation of the miniTn10'lacZ insertions in these strains. Morespecifically, genomic DNAs were digested with HindIII, an enzyme thatcuts once within miniTn10'lacZ (FIG. 1), and hybridized with pEH40 (FIG.2). The banding patterns revealed that each mutant had only onetransposon insertion but no additional pEH40 sequences. However, strainsNU229, NU230, and NU231 contained identical insertions (see lanes A, B,and C). These results were confirmed by a Southern hybridization usingEcoRI digested DNAs (data not shown). Thus, in this initial screening,six unique lacZ fusions were identified that appeared to beiron-regulated. of course, some of these strains may bear mutations inthe same gene or operon. Because the goal was to assess the role, ifany, for iron-repressed genes in L. pneumophila intracellular infection,rather than an exhaustive search for the total number of iron-regulatedgenes, these six were the subject of further investigations.

To quantitate the repression of these gene fusions by iron, liquidβ-galactosidase assays were performed on NU229, NU232, NU233, NU234 4,NU235, NU236. In two initial trials, all strains, except for NU233,produced less β-galactosidase when grown under high-iron conditions(FIG. 3A, P<0.0001, Student's t-test). NU229 and NU232 showed a 17 foldrepression, whereas NU234, NU235, and NU236 exhibited 2-3 fold drops inlacZ expression. Similar levels of repression have been seen in otheriron-regulated genes (e.g., E. coli sodA (2-3 fold) and Vibrioanguillarum fatA (14 fold)). Niederhoffer, et al., J. Bacteriol.172:1930-1938 (1990); Tolmasky, et al., J. Bacteriol. 176:213-220(1994). In subsequent trials that used increased amounts of DFX, agreater derepression was seen for NU229 and NU232. However, thephenotypes of NU234, NU235, and NU236 were different. In each case, thelevel of lacZ expression on standard BCYE agar appeared to increase(data not shown). Thus, β-galactosidase production in the presence ofEDDA was assessed to confirm the nature of iron-regulation within thesestrains (FIG. 3B). Like DFX, EDDA is best able to chelate Fe³⁺, havingan affinity of 10³⁴. Freedman, et al., Nature 179:1020-1021 (1957);Miles and Khimji, J. Med. Microbiol. 8:477-492 (1975). In theseexperiments, the levels of β-galactosidase production in NU234, NU235,and NU236 remained unresponsive to iron. The reason for these changesare not known, but it is assumed that compensatory mutations weresustained and maintained during passage and storage. However, the lacZfusions in NU229 and NU232 again demonstrated significant derepressionwhen grown on BCYE agar containing an iron chelator. Taken together,these experiments indicate that two mutants, NU229 and NU232, containstable iron-repressed 'lacZ fusions. The inactivated genes have beendesignated as frg for iron (fe)-repressed-gene.

2. Fur Repression of the L. pneumophila frq::lacZ Fusions

To begin to understand the mechanism of frg iron regulation, lacZexpression in L. pneumophila strains that differed only in Furproduction was assessed. Western blot analysis confirmed the earlierprediction that strain 130b expresses a protein that is cross-reactivewith and slightly smaller than E. coli Fur (data not shown). Hickey andCianciotto, Gene 143:117-121 (1994). Initially, it had been hoped thatcloned L. pneumophila fur and allelic exchange could be used toinsertionally inactivate the chromosomal fur genes. However, allattempts at this method were unsuccessful in strain 130b (data notshown). The inability to interrupt fur also has been seen in Neisseriagonorrhoeae, Neisseria menigiditis, P. aeruginosa, and V. anguillarum.Berish, et al., Infect. Immun. 61:4599-4606 (1993); Prince, et al., J.Bacteriol. 175:2589-2598 (1993); Thomas and Sparling, Molec. Microbiol.11:725-737 (1994); Tolmasky, et al. J. Bacteriol. 176:213-220 (1994). Asan alternate approach toward isolating Fur mutants, spontaneousmanganese-resistant (Mn^(r)) mutants were sought. Since Mn²⁺ and Fe²⁺can displace each other as co-factors, manganese resistance in E. coliis often achieved through fur mutations that permit increasedintracellular iron levels. Hantke, Mol. Gen. Genet. 210:135-139 (1987).The feasibility of this approach was tested in wild-type L. pneumophila.Mn^(r) legionellae were isolated on BCYE agar containing 0.4-0.6 mMMnCl₂ and examined for the loss of Fur by Western blot analysis (Table1). Whereas strains isolated on 0.4 mM manganese expressed full-sizedFur at normal levels, those obtained on higher concentrations ofmanganese either expressed a more rapidly migrating protein or anundetectable amount of Fur. The apparently truncated proteins wereapproximately 5-kDa smaller than Fur and appeared as faint bands on theimmunoblot. Introduction of cloned L. pneumophila fur into Mn^(r)strains restored manganese-sensitivity (data not shown). Therefore, asin P. aeruginosa, V. anguillarum, and V. cholerae, it is possible toisolate L. pneumophila Fur mutants by selecting for MnCl₂ resistance.Litwin, et al., J. Bacteriol. 174:1897-1903 (1992); Prince, et al., J.Bacteriol. 175:2589-2598 (1993); Tolmasky, et al., J. Bacteriol.176:213-220 (1994).

                  TABLE 1    ______________________________________    Manganese resistance and alterations in    Fur expression.sup.a                     Mn.sup.r #       #          Frequency  strains  full-   trun-                                           #          of Mn.sup.r                     tested for                              length  cated                                           absent     MnCl.sub.2!          CFU        Fur      Fur     Fur  Fur    ______________________________________    0.1 mM          8.5 × 10.sup.-1                     0         nd.sup.b                                      nd   nd    0.2 mM          5.0 × 10.sup.-4                     0        nd      nd   nd    0.4 mM          1.2 × 10.sup.-6                     2        2       0    0    0.5 mM          3.6 × 10.sup.-7                     10       3        1.sup.c                                           6    0.6 mM          3.6 × 10.sup.-7                     4         1.sup.c                                       2.sup.c                                           1    ______________________________________     .sup.a Bacteria were grown on BCYE media for 48 hours and then resuspende     in sterile water. Serial dilutions were then plated on BCYE agar     containing MnCl.sub.2 ranging from 0 to 0.6 mM.     .sup.b Not determined     .sup.c Crossreactive band on the Western blot was barely detectable.

Returning to the original goal of assessing Fur-regulation of frg,Mn^(r) mutants of NU229 and NU232 were derived on 0.6 mM MnCl₂ and thentested for the loss of Fur by Western Blot. Although a Fur null mutantof NU229 was readily obtained, all seven Mn^(r) derivatives of NU232tested still produced a full size Fur protein (data not shown). Todetermine if the frg::lacZ fusion in NU229 is Fur-repressed,quantitative β-galactosidase assays were performed using lysates fromMn^(s) and Mn^(r) bacteria grown on standard (high iron) BYCE agar(Table 2). In three trials, the Mn^(r) derivative displayed elevatedenzyme activity (P<0.001, Student's t-test), indicating that the loss ofFur allows for frg::lacz expression in the presence of high iron. Thedifferences in β-galactosidase production within the Fur⁻ populationslikely reflect reversion of the fur mutation since the bacteria used inthe assays were necessarily grown in the absence of manganese. Incontrast to Mn^(r) NU229, a representative Mn^(r) derivative of NU232did not display derepression of lacZ when it was grown on high ironmedia (data not shown), a result compatible with the presence offull-length Fur (see above). Strain NU229 became the focus of attentionsince it possessed a stable iron- and Fur-regulated lacZ fusion,designated frgA.

                  TABLE 2    ______________________________________    NU229 frgA: :lacz expression in the presence or    absence of Fur               β-galactosidase units.sup.a    Trial.sup.b  NU229 Fur.sup.+                           NU229 Fur.sup.-    ______________________________________    1            4.2 ± 0.4                           160.2 ± 20.1    2            2.4 ± 1.5                           320.5 ± 9.4    3            3.1 ± 0.3                           583.4 ± 10.0    ______________________________________     .sup.a Fur.sup.+ (Mn.sup.s) NU229 and Fur.sup.- (Mn.sup.r) NU229 were     grown on standard BCYE agar for 48 hours.     .sup.b For each trial, each strain was examined in triplicate.

3. Extracellular Growth of NU229.

Because many iron- and Fur-repressed genes are involved in iron-uptake,it was determined if the interruption of frgA affected extracellulargrowth, particularly under iron-stressed conditions. More specifically,strains 130b and NU229 were compared for their abilities to grow in CDMcontaining either 0, 4, 8, or 16 μM of added iron (FIG. 4). As expected,no significant growth of 130b was seen in the absence of iron and, asthe concentration of iron increased, the duration of the lag phasedecreased. Upon entering logarithmic growth, all 130b cultures reachedstationary phase within 48-72 hours. Although the final OD₆₆₀ achievedby the NU229 cultures was slightly lower than that reached by 130b, thegrowth characteristics of the mutant were remarkably similar towild-type (FIG. 4B-4D). Indeed, the mutant entered logarithmic growth atthe same time as the wild-type strain and had comparable growth rates.Similar growth characteristics for 130b and NU229 were also seen whenthese strains were grown in BYE (data not shown). Therefore, it wasconcluded that the interruption of frgA had minimal effects on theextracellular growth capacity and extracellular ironacquisition/assimilation functions of L. pneumophila. To furtherdemonstrate the latter point, we assessed the mutant's sensitivity toEDDA and its resistance to streptonigrin, an antibiotic that acts withinternal iron sources to inhibit bacterial growth. White and Yeowell,Biochem. Biophys. Res. Commun. 106:407-411 (1982). Toward this end,130b, NU229, and strains previously shown to be eitherstreptonigrin-resistant or EDDA-sensitive Pope, et al., Infect. Immun.64:629-36 (1996)! were plated on BCYE containing either 0.5 μg/ml of theantibiotic or 50-80 μM of the chelator. Both 130b and NU229 failed togrow on the streptonigrin medium and grew comparably in the presence ofEDDA (data not shown), indicating that there was no change in ironassimilation or storage in the frgA mutant under standard extracellulargrowth conditions.

4. Intracellular Infection by L. pneumophila frg Mutants

Because intracellular growth is the focal point of Legionellapathogenesis, the next step was to determine if NU229 was impaired ininfection of macrophages. Initially, ID₅₀ analyses were performed usingmacrophage-like U937 cells. In four separate experiments, strain NU229displayed a 3.2±1.4 fold increase in ID₅₀. More importantly, themonolayers infected with the mutant appeared to contain fewer bacteriathan those infected with the wild-type strain. Taken together, thesedata suggested that frgA has a role in infection in addition to beingiron- and Fur-regulated, Incidentally, NU232, NU234, and NU235 showed nochange in infectivity during these same ID₅₀ experiments, whereas NU236exhibited an 11.2±3.1 fold increase in ID₅₀ (95% confidence limits aredefined by ID₅₀ ratios of 5.0 and 17.4). To confirm that the infectivitydefect seen in NU229 was significant, U937 cell monolayers were infectedwith comparable numbers of 130b and NU229, and the levels ofintracellular bacteria were assessed at 0, 6, 12, 24, 48, and 72 hourspost-inoculation (FIG. 5A). At the first three timepoints, there were nodifferences in recovery between the wild-type and the mutant, indicatingthat NU229 is not impaired in entry or resistance to immediateintracellular killing. Beginning at 24 hours post-inoculation, however,significantly fewer bacteria were recovered from monolayers infectedwith NU229. Indeed, there were 9, 80, and 30 fold fewer bacteria at 24,48, and 72 hours post-inoculation, respectively. Similar results wereobtained in two subsequent experiments that assayed the number ofbacteria per monolayer at 72 hours post-inoculation (data not shown).Therefore, NU229 has a significant and reproducible defect inintracellular growth.

It was next investigated whether the reduced infectivity of NU229 wasdue to the inactivation of frgA rather than a spontaneous second-sitemutation. Thus, the mutant was remade by allelic exchange. Morespecifically, an approximately 12-kb PstI fragment containing frgA::lacZ(and Km^(r)) was isolated from NU229 and cloned into the PstI site insacB-containing pEA75. Following electroporation of the recombinantplasmid into strain 130b, six Km^(r), sucrose-resistant colonies wereobtained. All of these clones, when tested by Southern hybridization,contained the same DNA insertion as NU229 (data not shown). In addition,all of the clones produced higher levels of β-galactosidase when grownunder low-iron conditions, as did NU229. One strain, designated asNU229R, was further tested in U937 cells. NU229R demonstrated noinfectivity difference from the original NU229 on three separateoccasions, one of which is represented in FIG. 5A. The similaritiesbetween NU229 and NU229R indicate that the frgA interruption, not asecond-site mutation, caused the defect in intracellular growth.

5. Sequence Analysis of L. pneumophila frgA.

As the first step towards understanding the behavior of NU229, thenucleotide sequence of frgA was determined. Toward that end, the 12-kbPstI fragment containing frgA::lacZ was cloned into pSU2719 to yieldpEH44 (FIG. 6). To initiate sequence analysis using IS10 primers, thesubclones pEH45 and pEH46 (FIG. 6) were utilized. Subsequent sequencingreactions were performed with the pEH44 template DNA and a series ofcustom primers. To confirm the nucleotide sequence at the point oftransposon insertion, a plasmid (pEH65) containing the uninterrupted 3'end of frgA was isolated from a genomic library using probe A (see FIG.6) and then analyzed. FIG. 7 contains the sequence of the frgA openreading frame (ORF), as well as flanking regions SEQ ID NO: 4!, and thepredicted amino acid sequence SEQ ID NO;5!. The frgA ORF begins at anATG start codon and ends 1725 bp downstream at a TAA stop codon. ThisORF would encode a protein of 575 amino acids and 63.3 kDa. The L.pneumophila frgA ORF was preceded by a ribosome binding site within 10bp of the start codon, as well as a -10 and a -35 region that closelymatched the E. coli consensus. Rosenberg and Court, Annu. Rev. Genet.13:319-353 (1979). Most promoter regions of genes regulated by Furcontain at least one ironbox Calderwood and Mekalanos, J. Bacteriol.169:4759-4764 (1987); de Lorenzo, et al., J. Bacteriol. 169:2624-2630(1987)!, generally occurring within the -35 region to the -10 region,that represents the binding site(s) for Fur-Fe²⁺ repressors Litwin, etal., J. Bacteriol. 174:1897-1903 (1992)!. The L. pneumophila frgApromoter region contained two overlapping ironboxes that had 4-5mismatches, as compared to the E. coli consensus sequence, 5'-GATAATGATAATCATTATC SEQ ID NO:47! (FIG. 7). Calderwood, et al., J. Bacteriol.169:4759-4764 (1987); de Lorenzo, et al., J. Bacteriol. 169:2624-2630(1987). This finding supports our earlier conclusion that frgA is iron-and Fur-regulated.

Database searches using the predicted amino acid sequence of frgA showedthat it shares homology with two proteins, IucA and IucC, encoded by thefirst and third genes in the Fur-repressed E. coli aerobactin operon(FIG. 8; SEQ ID NOS: 6-44). Martinez, et al., J. Mol. Biol. 238:288-293(1994). In the third step of the siderophore's biosynthesis, IucAcatalyzes the addition of N.sup.ε -acetyl-N.sup.ε -hydroxylysine tocitric acid to yield the intermediate N.sup.α -citryl-N.sup.ε-acetyl-N.sup.ε -hydroxylysine. Subsequently, IucC adds another N.sup.ε-acetyl-N.sup.ε -hydroxylysine moiety to the intermediate to formaerobactin. de Lorenzo and Neilands, J. Bacteriol. 167:350-5 (1986). Ina previous report by de Lorenzo, et al. Martinez, et al., J. Mol. Biol.238:288-293 (1994)!, a 21% identity and 47% similarity between IucA andIucC was recognized as significant. While IucA and IucC catalyze similarreactions and have structural homology, they are not interchangeable (deLorenzo and Neilands, J. Bacteriol. 167:350-5 (1986)). The overallidentity and similarity between L. pneumophila FrgA and E. coli IucAwere 15.7% and 26.1% respectively. Furthermore, FrgA had an 18.3%identify and a 32.6% similarity to IucC. BLASTX results from the NCBIspecified the three regions of greatest homology between FrgA and the E.coli proteins. The three boxes in FIG. 8 denote these regions for IucA,and the BLASTX identity:similarity results were 23%:51%, 29%:45%, and31%:46%, respectively. The three shaded areas denote these regions forIucC with BLASTX identify:similarity results of 21%:47%, 45%:75%,32%:51% respectively. It is also worth noting that FrgA is comparable insize to both IucC and IucA; indeed, it is predicted to have the samenumber of amino acids as IucA (FIG. 8). Taken together, these datasuggest that frgA encodes an IucA- or IucC-like protein. Sequencesupstream and downstream of frgA, however, did not reveal any ORFs thatcould be a part of an iuc-like operon (FIG. 7 and data not shown).

6. Complementation of the frgA Mutation.

To determine if the infectivity defect in NU229 is due to the loss ofFrgA or due to a polar effect (i.e., a block in transcription of a genedownstream of frgA), efforts were made to restore full infectivity tothe mutant by introducing into it an intact plasmid-encoded frgA. First,using a frgA specific probe (see probe B in FIG. 6), the complete genewas isolated on plasmid pEH74 from our genomic library. Next, frgA wassubcloned on a 2886 bp KpnI-XhoI fragment into the vector pSU2719 toyield pEH75. Although pEH75 contains 1093 bp upstream of the frgA startcodon, it does not contain any ORFs that would be transcribed in thesame direction as frgA. More importantly, pEH75 does not contain anydownstream ORFs since the XhoI site is only 62 bp downstream from thefrgA stop codon (FIG. 7). Finally, pEH75 was electroporated into NU229,and transformants were obtained on BCYE containing kanamycin andchloramphenicol. To have controls for the infectivity studies, NU229 and130b containing the Cm^(r) pSU2719 vector were isolated. In threeseparate experiments, one of which is depicted in FIG. 5B, strain NU229(pEH75) exhibited intracellular growth characteristics that werecomparable to 130b. This result demonstrates that the presence of frgAalone was sufficient to restore infectivity to NU229, and that this geneis required for optimal intracellular infection.

7. Distribution of frgA Among L. pneumophila Strains and LegionellaSpecies

To determine if frgA is conserved among different L. pneumophila strainsand Legionella species, southern hybridization analyses were performedusing probe B (FIG. 6). The frgA specific probe hybridized under highstringency conditions to DNAs from the eleven strains of L. pneumophilathat were tested (i.e., representatives of serogroups 1-4 and 6-14)(FIG. 9). However, the probe did not hybridize, even under lowstringency conditions, with DNAs from L. birminghamensis, L. dumoffii,L. erytha, L. gormanii, L. feeleii, L. hackeliae, L. israelensis, L.jamestowniensis, L. longbeachae, L. micdadei, L. oakridgensis, L.parisiensis, L. sainthelensi, L. santicrusis, L. spiritensis, and L.tucsoniensis (FIG. 9). The strains tested represent both clinical andenvironmental isolates. These results indicate that frgA is conservedwithin, and is specific to, strains of L. pneumophila.

C. Discussion

The identification of genes whose expression is repressed by Fur inresponse to iron availability may lead to the isolation of virulencedeterminants for two central reasons. First, genes encodingiron-acquisition systems can themselves be essential for bacterialsurvival within mammalian hosts. Examples of these are the ferrous ironuptake systems and the aerobactin operon of pathogenic E. coli. Kammler,et al., J. Bacteriol. 175:6212-6219 (1993); Payne, Trends Microbiol.1:66-69 (1993); Valvano and Crosa, Infect Immun. 46:159-167 (1984).Second, factors not directly involved in iron assimilation can berepressed by Fur. Several examples of these are the Shiga-like toxin andsuperoxide dismutase from E. coli, a hemolysin and the outer membraneIrgA protein from V. cholerae, and exotoxin A from P. aeruginosa.Calderwood and Mekalanos, J. Bacteriol. 169:4759-4764 (1987); Goldberg,et al., Proc. Natl. Acad. Sci USA 88:1125-1129 (1991); Litwin, et al.,J. Bacteriol. 174:1897-1903 (1992); Niederhoffer, et al., J. Bacteriol.172:1930-1938 (1990); Prince, et al., J. Bacteriol. 175:2589-2598(1993). The majority of information on iron- and Fur-repressed genescomes from studies on either extracellular pathogens or facultativeintracellular parasites of the gastrointestinal tract. Litwin andCalderwood, Clin. Microbiol. Rev. 6:137-149 (1993).

The data indicated that L. pneumophila Fur is structurally andantigenically similar to E. coli Fur, but the Legionella fur gene,unlike Escherichia fur, appears to be essential for bacterial viability,as seen by the inability to insertionally inactivate it. A complete lossof Fur in L. pneumophila, as well as in N. meningiditis, N. gonorrhoeae,P. aeruginosa, and V. anguillarum, probably increases the levels of ironto a point that is bactericidal under aerobic growth conditions. Berish,et al., Infect. Immun. 61:4599-4606 (1993); Prince, et al., J.Bacteriol. 175:2589-2598 (1993); Thomas and Sparling, Molec. Microbiol.11:725-737 (1994); Tolmasky, et al., J. Bacteriol. 176:213-220 (1994);Touati, et al., J. Bacteriol. 177:2305-2314 (1995). To studyFur-regulated genes, spontaneous Mn^(r) mutants were used. Mn^(r)strains of V. anguillarum and V. cholerae have been shown to bear pointmutations in fur, generally causing changes in the amino-terminal halfof Fur. Lam, et al., J. Bacteriol. 176:5108-5115 (1994); Wertheimer, etal., J. Bacteriol 176:5116-5122 (1994). Since minimal levels of Furactivity are likely present in our Mn^(r) mutants, it may not be able toidentify genes whose expression is regulated only slightly by Fur.Nevertheless, the ability to generate fur mutants and effectivelyintroduce 'lacZ into virulent legionellae allowed study of the ironregulation of five L. pneumophila (frg) loci.

Stable iron regulation was noted in two of the five strains, NU232 andNU229. The frg (frgB) in strain NU232 was repressed 17 fold in responseto iron. However, its Fur regulation could not be assessed because anNU232 Fur⁻¹ derivative could not be obtained. An initial attempt atdemonstrating Fur repression of frgB in E. coli also provedinconclusive, i.e., when cloned along with 3 kb of upstream DNA,frgB-lacZ was expressed to a similar degree in Fur⁺ and Fur⁻ strains(data not shown). The basis for the lethality of the fur-frgB doublemutation is unclear, but it likely involves toxic levels ofintracellular iron associated with the fur mutation. This result mayprovide a clue to frgB function. For example, the inability of the frgBmutation to offset Fur derepression suggests that it is not involved iniron-uptake. Instead, it may encode an iron-storage protein, such asbacterioferritin or the ferritin H-like protein, whose absence wouldfacilitate iron overload. Andrews, et al., J. Bacteriol. 171:3940-3947(1989); Izuhara, et al., Mol. Gen. Genet. 225:510-513 (1991).Alternately, it may express a protein, such as a DNA repair enzyme,superoxide dismutase, or peroxidase, involved in the protection of DNAdamage from iron-catalyzed oxygen radicals. Touati, et al., J.Bacteriol. 177:2305-2314 (1995). The frgA gene in strain NU229 had asimilar level of iron-regulation as frgB, but was repressed by Fur.Since a fur mutation in NU229 could be isolated, frgA is more likely tobe involved in iron uptake than the frgB. The level of derepressionassociated with the loss of Fur was greater than that achieved by growthunder low-iron conditions. A likely explanation for this result is thatsome Fur dimers were still bound to promoter regions at the levels ofiron chelator that were tested. Assessment of lacZ expression underconditions of greater iron limitation was attempted. However, anincreased concentration of the chelator caused an inhibition of growthby both wild-type and mutant strains, prohibiting any furtherexamination of specific gene expression. Regardless, thecharacterization of NU229 demonstrates that iron regulation in L.pneumophila is explained, at least in part, by Fur-mediated repression.

Although essentially unaltered in its extracellular growth capability,NU229 was significantly impaired in intracellular growth. Despite anapparently normal capacity to enter into host cells and to resistmacrophage bacteriocidal elements, the mutant replicated in a mannerthat yielded significantly fewer progeny than wild-type in a 72 hourtime period. A comparison of macrophage growth kinetics suggests thatNU229 is distinct from all other L. pneumophila mutants (i.e., mip, icm,dot, and ira mutants) found to be impaired for intracellularreplication. Berger, et al., Molec. Microbiol. 14:809-22 (1994); Brand,et al., Molec. Microbiol. 14:797-808 (1994); Cianciotto, et al., InfectImmun. 57:1255-1262 (1989); Pope, et al., Infect. Immun. 64:629-36(1996). Complementation analysis further demonstrated that theinfectivity defect of NU229 was due directly to the inactivation offrgA. Thus, frgA can be added to the small but growing list of L.pneumophila infectivity genes. Furthermore, the demonstration that aniron- and Fur-regulated gene can promote intracellular growth supportsthe notion that the phagosomal compartment is a low-iron environment.Pope, et al., Infect. Immun. 64:629-36 (1996); Portillo, et al., Mol.Microbiol. 6:3289-3297 (1992).

The L. pneumophila FrgA protein was similar to IucA and IucC in theaerobactin iron-uptake system of E. coli. A comparison of IucA and IucCsequences disclosed three hydrophilic domains of greater than 57%similarity which are thought to contain the active centers of theseenzymes. Martinez, et al., J. Mol. Biol. 238:288-293 (1994).Interestingly, the regions of highest homology between FrgA and the Iucproteins overlap these sequences. Thus, the 21-45% amino acid identitiesthat exist between FrgA and IucA/C may reflect a common function. In apotentially analogous situation, the carboxyl-terminals of L.pneumophila Mip and the FK506-binding protein of Neurospora crassa share39% of their sequence and still possess similar peptidyl-prolylcis/trans isomerase activity. Fischer, et al., Molec. Microbiol.6:1375-1383 (1992); Tropschug, et al., Nature 346:674-677 (1990). Thediscovery that frgA has homology with genes intimately involved insiderophore biosynthesis was exciting but also quite unexpected forseveral reasons. First, the absence of a aerobactin-like siderophore inL. pneumophila strains was demonstrated by the Csaky assay, whichidentifies hydroxymate siderophores due to their chemical structure.Reeves, et al., J. Bacteriol. 154:324-329 (1983). Second, theChrom-Azurol S (CAS) assay, a procedure which measures chelatingactivity independent of structure, failed to detect siderophores inculture supernatants of strain 130b. Goldoni, et al., J. Med. Microbiol.34:113-8 (1991); Liles and Cianciotto, Infect Immun. 64:1873-1875(1996). Third, in a classic bioassay, concentrated supernatants from oneiron-depleted L. pneumophila culture did not stimulate the growth of asecond iron-starved culture. Reeves, et al., J. Bacteriol. 154:324-329(1983). Finally, NU229 was not sensitive to iron limitation and was notresistant to streptonigrin when grown on bacteriological media. On theother hand, the importance of frgA for growth within the iron-limitingenvironment of a macrophage and its iron-dependent regulation by Fursuggests a role in iron-acquisition. These data can be rationalized inone of two ways. Either FrgA catalyzes a reaction akin to the onesmediated by IucA and IucC, but does not result in the production of a"classic" siderophore or, along with unlinked loci, frgA is involved inthe production of an iron chelator prevalent in the intracellularenvironment. The absence of Fe³⁺ -chelating activity in L. pneumophilasupernatants, despite frgA expression in low-iron bacteriological media,would suggest that the production of this putative siderophore is notsolely responsive to iron and Fur fluctuations. Clearly, the role offrgA in iron acquisition is yet to be elucidated; however, its sequencehas caused us to once again reexamine our view of L. pneumophila andsiderophores.

The frgA gene was interesting not only for its role in macrophageinfection and its sequence homologies, but also for its uniquedistribution among legionellae. Southern hybridization analysisindicated that frgA sequences are limited to the L. pneumophila species.Nearly all previously identified L. pneumophila genes, including flaA,fur, htp, lly, mip, ompS, and pplA (pal), have had homologs in all orvirtually all other Legionella species. Bender, et al., Infect. Immun.59:3333-6 (1991); Cianciotto, et al., Infect Immun. 58:2912-2918 (1990);Heuner, et al., Infect. Immun. 63:2499-507 (1995); Hickey andCianciotto, Gene 143:117-121 (1994); Hoffman, et al., Infect. Immun.57:1731-1739 (1989); Hoffman, et al., J. Bacteriol. 174:914-920 (1992);Ott, et al., Microb. Pathog. 11:357-65 (1991). The other exceptionalgene beside frgA is hbp, whose isolation and identification is describedin Example 2. See also O'C.onnell, et al., Infect. Immun. 64:842-848(1996). The absence of frgA among the sixteen Legionella speciesexamined signals that the gene can be used as a diagnostic reagent. Themip primers used for the PCR detection of L. pneumophila within waterand clinical samples have, on several instances, amplified DNAs fromother Legionella species. Atlas, et al., App. Environ Microbiol.61:1208-1213 (1995); Cianciotto and Fields, Proc. Natl. Acad. Sci. USA89:5188-5191 (1992); Jaulhac, et al., J. Clin Microbiol 30:920-924(1992); Kessler, et al., J. Clin. Microbiol. 31:3325-3328 (1993);Lindsay, et al., J. Clin. Microbiol. 32:3068-3069 (1994). Thus, frgAprimers, even more so than hbp primers, should enhance the specificityof L. pneumophila detection protocols (see Example 2).

Example 2 Cloning and Mutation of L. pneumophila hbp Genes

A. Materials and Methods

1. BACTERIAL STRAINS AND MEDIA. Virulent L. pneumophila serogroup 1strains Wadsworth 130b and Philadelphia 1, serogroup 8 strain Concord 3,and serogroup 13 strain B2A3105 were previously described. Cianciotto,et al., Mol. Biol. Med. 6:490 (1981). Strains of Legionella erthyra, L.feeleii, L. hackeliae, L. longbeachae, L. micdadei, L. moravica, L.sainticrucis, and L. spiritensis were also included in this study.Cianciotto, et al., Mol. Biol. Med. 6:490 (1981). Like L. pneumophila,the species L. feeleii, L. hackeliae, L. longbeachae, and L. micdadeihave been associated with human disease. Generally, the legionellae weregrown on standard BCYE agar plates for 48 h at 37° C., and, whenappropriate, 3 μg of chloramphenicol per ml, 25 μg of kanamycin per ml,or 5% (wt/vol) sucrose was added to the medium. For experiments thatassessed hemin utilization and binding, a slightly modified form ofyeast extract phosphate (YP) medium was employed as the base medium.Armon, and Payment, J. Microbiol. Methods 11:65-71 (1990); Johnson, etal., J. Clin. Microbiol. 15:342-344 (1982). This medium contained, as asupplement, 250 mg of ferric pyrophosphate (PP_(i)) and 400 mg ofcysteine per liter. The chemically defined medium (CDM) described byReeves et al. was employed to further assess the ability of Legionellaspecies to use hemin as an iron source. Reeves, et al., J. Clin.Microbiol. 13:688-695 (1981). BYE broth cultures were washed in CDM andthen inoculated into acid-washed, 125-ml flasks containing 25 ml of CDM.The resultant cell suspensions were incubated at 37° C. with agitation.

E. coli HB101 served as the host for recombinant plasmids. Currentprotocols in molecular biology (Ausubel, et al., eds., 1987). It wasmaintained on Luria-Bertani agar medium containing either 30 μg ofchloramphenicol, 50 μg of kanamycin, or 50 μg of ampicillin per ml.Current protocols in molecular biology (Ausubel, et al., eds., 1987).Recombinant E. coli cells were also grown on M9CA-salts agarsupplemented with either 100 μg of hemin or Congo red per ml. Hanson andHansen, Mol. Microbiol. 5:267-278 (1991).

Unless otherwise noted, chemicals were obtained from Sigma Chemical Co.,St. Louis, Mo.

2. Plasmids and a Genomic Library. Plasmids pBR322 and pUC18 were usedas cloning vehicles. Current protocols in molecular biology (Ausubel, etal., eds., 1987). Another ColE1 replicon, pBOC20, was used in theallelic exchange protocol. This plasmid represents the multicloning sitefrom PHXK cloned into pEA75. O'C.onnell et al., Infect. Immun.63:2840-2845 (1995). Importantly, it contains a selectablechloramphenicol resistance (Cm^(r)) marker and the counterselectablesacB. The vector pNK2794 served as the source for a 1.7-kb BamHIfragment which contains a kanamycin resistance (Km^(r)) gene. O'C.onnellet al., Infect. Immun. 63:2840-2845 (1995). Finally, the mip-containingplasmid pSMJ31.42 was used as a probe in Southern hybridizations.Engleberg, et al., Infect. Immun. 57:1263-1270 (1989). Plasmids wereisolated from E. coli by the alkaline lysis procedure. Current protocolsin molecular biology (Ausubel, et al., eds., 1987). The genomic libraryused in this study was derived from L. pneumophila 130b and consisted of3-6 kb Sau3A fragments cloned into pBR322. Hickey and Cianciotto, Gene143:117-121 (1994).

3. Electroporation and Allelic-exchange Mutagenesis. Plasmids wereintroduced into L. pneumophila by electroporation. Cianciotto andFields, Proc. Natl. Acad. Sci USA 89:5188-5191 (1992). The procedure forallelic exchange with ColE1 vectors containing counterselectable markershas been previously described. Cianciotto, et al., FEMS Microbiol. Lett.56:203-208 (1988); O'C.onnell, et al., Infect. Immun. 63:2840-2845(1995). Using this protocol, we achieved insertional inactivation of hbpwithin a strain that had been passaged six times on BCYE agar plates.

4. Northern (RNA) and Southern Hybridizations. Whole-cell RNAs and DNAswere extracted from Legionella strains as described previously.Cianciotto, et al., FEMS Microbiol. Lett. 56:203-208 (1988); Engleberg,et al., Infect. Immun. 57:1263-1270 (1989). Northern hybridizations wereperformed by standard protocols. Current protocols in molecular biology(Ausubel, et al., eds., 1987). RNAs were electrophoresed through a gelthat contained 1% agarose and 2.2M formaldehyde and that was bathed in1× morpholinepropanesulfonic acid (MOPS) buffer. The sizes of themolecular weight standards that were used to estimate the lengths ofmRNA species were 9.5, 7.5, 4.4, 2.4, 1.4, and 0.24 kb (Gibco-BRL,Gaithersburg, Md.). Southern hybridizations were performed under high-and low-stringency conditions which permit approximately 10 and 30% bpmismatching, respectively. Current protocols in molecular biology(Ausubel, et al., eds., 1987); Cianciotto, et al., Infect. Immun.58:2912-2918 (1990). Probes consisted of both plasmids and gel-isolatedrestriction fragments which were radiolabeled with ³² P by using arandom primer labeling kit (Gibco-BRL).

5. DNA Sequence Analysis. Cloned L. pneumophila DNA was sequenced fromdouble-stranded plasmids by the dideoxy chain-termination method with ³⁵S-dATP and Sequenase (Amersham, Arlington Heights, Ill.). Currentprotocols in molecular biology (Ausubel, et al., eds., 1987). Initially,M13-based primers were used to sequence DNAs cloned into pUC18; however,custom 20-bp oligodeoxyribonucleotide primers were used in subsequentreactions. The unique primers were prepared by the NorthwesternUniversity Biotechnology Center with an Applied Biosystems DNAsynthesizer. Sequencing reactions were performed according to themanufacturer's protocols. Nucleotide sequences were analyzed with PCGENE(IntelliGenetics), and homology searches were conducted through GenBankat the NCBI.

6. Liquid Hemin-binding Assay. To quantitate the ability of L.pneumophila and E. coli strains to bind hemin, the standard liquidhemin-binding assay was employed. Daskaleros and Payne, Infect. Immun.55:1393-1398 (1987); Deneer and Potter, Infect. Immun. 57:798-804(1989); Genco, et al., Infect. Immun. 62:2885-2892 (1994); Hanson andHansen, Mol. Microbiol. 5:267-278 (1991); Kay, et al., J. Bacteriol.164:1332-1336 (1985). Prior to exposure to hemin, the legionellae weregrown for 48 h on agar media and then subjected to a series of washes.First, the bacteria were harvested from plates into 40 ml of distilledwater, achieving an optical density at 660 nm (OD₆₆₀) of approximately1.5. Then, after centrifugation of the cell suspension for 10 min at4,500×g, the pellet was dissolved in 15 ml of 0.1M Tris (pH 8.0).Subsequent to a second centrifugation step, the bacteria wereresuspended for the last time in 15 ml of BYE broth. After removal of0.1 ml from the final cell suspension for a CFU determination, 1-mlaliquots were placed into 1.5-ml Microfuge tubes and brought toconcentrations of either 5, 10, 15, or 20 μg of hemin per ml byadditions from a freshly prepared 1-mg/ml stock in BYE. Thebacterium-hemin mixture was then rotated at 37° C. After a 1-hincubation, the cell suspension was centrifuged for 2 min at 8,000×g.Finally, 0.75 ml of the supernatant was examined, along with theappropriate hemin-BYE control, for its A₄₀₀. As always, the extent ofhemin binding was a reflection of the reduction in the OD₄₀₀. E. colistrains were grown and assayed in M9CA salts broth.

7. Intracellular Infection of U937 Cells and Amoebae by L. pneumophila.U937 cell monolayers were prepared and infected with L. pneumophila aspreviously described. Cianciotto, et al., Infect. Immun. 57:1255-1262(1989); O'C.onnell, et al., Infect. Immun. 63:2840-2845 (1995);Pearlman, et al., Microb. Pathog. 5:87-95 (1988). Following inoculation,the monolayers were incubated for 2 h to permit bacterial uptake andwere then vigorously washed to remove unattached bacteria. The infectedmonolayers were incubated at 37° C. in RPMI medium supplemented with 10%fetal bovine serum. To assess the relative infectivity of strains forU937 cells, 50% infective doses were determined after 72 h ofincubation. Cianciotto, et al., Infect. Immun. 57:1255-1262 (1989). Tomonitor intracellular growth rates, replicate monolayers were inoculatedwith approximately 10⁶ bacteria, incubated for various times, and thenlysed. Cianciotto, et al., Infect. Immun. 57:1255-1262 (1989);O'C.onnell, et al., Infect. Immun. 63:2840-2845 (1995). Tenfold serialdilutions of the lysates were plated on BCYE agar, and the resulting CFUwere used to calculate the corresponding numbers of bacteria permonolayer. In some experiments, the U937 cells were treated before andafter infection with 7 μM DFX. DFX inhibits Legionella replicationwithin macrophages by reducing intracellular iron availability. Byrd andHorwitz, J. Clin. Invest. 83:1457-1465 (1989); Gebran, et al., Infect.Immun. 62:564-568 (1994); Pope, et al., Infect. Immun. 64:629-636(1996).

Intracellular infection of the freshwater amoeba Hartmannellavermiformis was performed as previously described. Cianciotto andFields, Proc. Natl. Acad. Sci USA 89:5188-5191 (1992); King, et al.,Infect. Immun. 59:758-763 (1991). Briefly, replicate Hartmannellacultures containing 10⁵ amoebae were infected with 10³ CFU, and aftervarious incubation periods, the numbers of legionellae within thecocultures were determined by plating aliquots on BCYE medium.

8. Nucleotide Sequence Accession Number. The hbp sequence has beendeposited in the GenBank database at the NCBI under accession numberU4338.

B. Results

1. Hemin Utilization and Binding by L. pneumophila. Since the ability ofhemin to enhance Legionella growth is most manifest on YP agar platesJohnson, et al., J. Clin. Microbiol. 15:342-344 (1992)!, the behavior ofwild-type strain 130b on this medium was examined. Initially, two keyobservations of the previous study were confirmed. First, the number ofCFU recoverable on YP agar was 3% of that recoverable on BCYE (Table 3below). Second, the addition of hemin to the medium increased the CFU bynearly 10-fold (Table 3). However, to highlight more clearly the role ofhemin in Legionella physiology, the growth of strain 130b on YP mediathat were lacking Fe³⁺ supplements and that were thus nonpermissive(Table 3) was examined. In six separate experiments (two of which appearin Table 3), the addition of 30 μM hemin completely restored the abilityof strain 130b to form colonies on low-iron YP medium. Importantly, anequimolar amount of protoporphyrin IX could not substitute for hemin.Taken together, these data suggest that hemin can be an iron source forL. pneumophila. To support this idea, the ability of hemin to replaceferric iron in a CDM was examined. The omission of ferric salts from theCDM prevented the growth of strain 130b, confirming that iron isessential for L. pneumophila replication. The addition of 6.25, 12.5, or25 μM Fe³⁺ to the medium fully restored bacterial growth (data notshown). More importantly, growth of 130b was supported by Fe³⁺ -free CDMthat had been supplemented with hemin. Whereas the addition of 6.25 μMhemin yielded 75 to 90% maximal growth, the addition of 12.5 or 20 μMhemin promoted full replication (data not shown). Since 6.25 μM Fe³⁺supported better growth than did an equimolar amount of heme-iron, it issuspected that ferric iron is the more effective iron source for thelegionellae. Nevertheless, the data demonstrated that hemin can be thesole iron source for L. pneumophila.

                  TABLE 3    ______________________________________    Plating efficiency of L. pneumophila 130b on YP media                   Average no. of                   CFU/ml recovered.sup.b    Agar medium.sup.a                     Expt 1   Expt 2    ______________________________________    BCYE             1.7 × 10.sup.8                              1.6 × 10.sup.8    YP               5.5 × 10.sup.6                              ND    YP + Hemin       4.7 × 10.sup.7                              ND    YP - Fe          <10.sup.2c                              <10.sup.1c    YP - Fe + hemin  3.9 × 10.sup.7                              2.1 × 10.sup.7    YP - Fe + PP     ND       <10.sup.1c    ______________________________________     .sup.a YP + hemin, YP supplemented with 30 μM hemin; YP - Fe, YP     lacking its ferric PP.sub.i supplement; YP - Fe + hemin, YP lacking ferri     PP.sub.i but supplemented with 30 μM hemin; YP - Fe + PP, YP lacking     ferric PP.sub.i but supplemented with 30 μM protoporphyrin IX.     .sup.b Bacteria were grown on BCYE agar plates for 48 h, resuspended in     distilled H.sub.2 O to an OD.sub.660 of approximately 0.3, and then plate     in triplicate for determinations of the numbers of CFU on the indicated     media. ND, not determined.     .sup.c No CFU recovered.

To substantiate the idea that L. pneumophila can directly utilize hemecompounds, we assayed strain 130b for its ability to bind hemin.Following growth on YP-minus-Fe-plus-hemin (Table 3) media, L.pneumophila consistently bound 50 to 60% of the added hemin (see FIG.10). This level of hemin binding was 10-fold greater than that of E.coli HB101 (see below). Legionellae harvested from BCYE agar plates alsobound appreciable amounts of hemin, indicating that L. pneumophila heminbinding was not a peculiarity of growth on YP media (FIG. 10).Interestingly, however, these bacteria adsorbed noticeably less of thecompound that did those obtained from the hemin-containing YP agar.Taken together, these initial experiments predicted that L. pneumophilahad surface structures (proteins) which promote hemin acquisition.

2. Identification of a L. pneumophila Gene That Promotes Hemin Binding.To identify L. pneumophila proteins involved in hemin acquisition, agenomic library was screened for a locus that could confer upon E. coliHB101 the ability to bind hemin. Using an approach which recently led tothe characterization of a hemin-binding membrane protein of H.influenzae Hanson and Hansen, Mol. Microbiol. 5:267-278 (1991)!,recombinant bacteria that appeared brown on M9CA salts agar platescontaining 0.01% hemin were sought. Since bacteria and proteins thatspecifically interact with hemin often bind Congo red Daskaleros andPayne, Infect. Immun. 55:1393-1398 (1987); Deneer and Potter, Infect.Immun. 57:798-804 (1989); Genco, et al., Infect. Immun. 62:2885-2892(1994); Kay, et al., J. Bacteriol. 164:1332-1336 (1985); Stugard, etal., Infect. Immun. 57:3534-3539 (1989)!, screening was for clones thatwere also colored on media containing that dye. Three pigmentedrecombinant strains were obtained. Importantly, the plasmids pEH1, pEH2,and pBOC3, which were isolated from these clones, conferred pigmentationupon retransformation into HB101. To confirm that HB101(pEH1),HB101(pEH2), and HB101(pBOC3) had enhanced hemin-binding activity, theirabilities to remove hemin from solution were assessed. Indeed, all threeclones bound about 60% more hemin than did HB101 (pBR322) (see FIG.11A). Restriction enzyme digestion analyses and Southern hybridizationsindicated that pEH1, pEH2, and pBOC3 were overlapping and contained L.pneumophila 130b DNA (see FIG. 12).

Subcloning mapped the locus responsible for hemin binding to a 1.1-kbSacI-AflII fragment (FIG. 12). DNA sequence analysis indicated that thisregion of the L. pneumophila chromosome contained one intact openreading frame (ORF). A Km^(r) insertion into the HincII site of pEH12abolished pigmentation in recombinant E. coli, confirming that this ORFis required for hemin binding (FIG. 12). The ORF is designated as hbpfor hemin binding promotion. (Note that in a preliminary report the ORFhad been referred to as heb for hemin binding. O'C.onnell andCianciotto, Abstr. B-7, p. 29, in Abstracts of the 94th General Meetingof the American Society for Microbiology 1994 (American Society forMicrobiology, Washington, D.C. 1994). The hbp ORF was predicted toencode a protein (Hbp) of 141 amino acids and 15.5 kDa. Interestingly,Hbp contained a 22-residue signal sequence (see Chart A below),indicating that it may be secreted in E. coli and L. pneumophila.Database searches failed to reveal significant degrees of homologybetween Hbp and known proteins.

The hbp ORF was preceded by a ribosome-binding site within 13 bp of theinitiation codon (Chart A). Further upstream, there existed twopotential -10 regions. The first of these putative promoter regionscontained several sets of sequences that had homology with Fur bindingsites. Hickey and Cianciotto, Gene 143:117-121 (1994). The presence ofironboxes suggested that hbp is regulated by L. pneumophila Fur andintracellular iron levels. Hickey and Cianciotto, Gene 143:117-121(1994). Finally, the presence of a potential transcription terminatorshortly after the Hbp stop codon suggested that hbp is transcribed as amonocistronic message (Chart A). To confirm this idea, Northern blotanalysis was performed using RNA isolated from strain 130b and, as aprobe, the hbp-containing NdeI-EcoRV fragment of pEH12 (FIG. 12). L.pneumophila expressed a single hbp-hybridizing transcript (data notshown). Importantly, the length of that mRNA was estimated to be 440bases, a size which is compatible with that of the hbp ORF.

Chart A gives the nucleotide sequence of the hbp region SEQ ID NO:45!and the predicted amino acid sequence of Hbp SEQ ID NO:46!. Potential-10 regions for the hbp promoter are in boldface lowercase type, and thearea containing possible iron boxes is underlined. The locations of keyrestriction enzyme recognition sites, the ribosome binding site, and thetranscription terminator are also clearly indicated. The putative signalsequence for Hbp is in boldface uppercase type.

    CHART A                                       Nde I    gaaaaatatccttataaatatgaattagccattgcatatggcaaaagataatctgaaaca   60    attcccgacaacatccttttaaaacggttgcaactgaaaattcaacttgttagtcttttg  120                              RBS                        Hinc II    aagatttttaactgctaagtaacaaggagaagtaacATGATGTTGAAAACCCAGTTGACT  180                                         M  M  L  K  T  Q  L  T     8    GCTTTTATCGGTGCTGTAATCTTGGCTGGCTCTTCTTTAGCAAATCCAATAAAACCTGAG  240     A  F  I  G  A  V  I  L  A  G  S  S  L  A  N  P  I  K  P  E    28    GTATGCCCCAGTGTACCCTCTATTCAATCGGAAGGAATGTCCATGTCTTCTGAAATTTTG  300     V  C  P  S  V  P  S  I  Q  S  E  G  M  S  M  S  S  E  I  L    48    GAGGGCATGTACATCACCTATAATTTAAGTCATTACAATACCAGTTCAAGCTGGGTGTTT  360     E  G  M  Y  I  T  Y  N  L  S  H  Y  N  T  S  S  S  W  V  F    68    ATTGTAGGGCCAATCGCAGCTGAAAATGATGATATGGCATTGGCAGAAAGCAATAAATTA  420     I  V  G  P  I  A  A  E  N  D  D  M  A  L  A  E  S  N  K  L    88    CTTTCAACCATGTCAGGGTCTCCCCATCCGGAAGATGATGGAGAAGGCAATTGGATATGT  480     L  S  T  M  S  G  S  P  H  P  E  D  D  G  E  G  N  W  I  C   108    CAGTATACGACCAAATCCAAAGATATTATTGCATTTGCCATAGAAGCAGATGATATGCTT  540     Q  Y  T  T  K  S  K  D  I  I  A  F  A  I  E  A  D  D  M  L   128                        Eco RV    TCTCCATTGAAAATGATGAGATATCTCAGAACAATCCGCTGATAAGTGGATAACACCTGC  600                         141 L  R  T  I  R                                                    Sac I    ACGGACACGGAAGCATGATTTGCTTCCGTGATTTAACGAATAGTTAAGAGCTC         653                                             SEQ ID NOS: 45 & 46!

3. Construction and Characterization of an L. pneumophila hbp Mutant. Toultimately determine whether hbp promotes hemin acquisition by L.pneumophila, an hbp mutant was isolated. Specifically, allelic exchangewas employed to insertionally inactivate the hbp gene within strain130b. The plasmid used for this procedure, pBOC22, was constructed intwo steps. First, a blunt-end 1.7-kb fragment containing Km^(r) wasintroduced into the HincII site of pEH12 to yield pBOC21. As notedabove, this DNA insertion into hbp abolished hemin binding inrecombinant E. coli. Second, the mutated gene was transferred on aSacI-BamHI fragment into the Cm^(r) and sacB-containing pBOC20. As thenext step toward the construction of the mutant, pBOC22 waselectroplated into strain 130b, and the transformation mixture wasplated onto BCYE agar containing both kanamycin and sucrose. Bysimultaneously selecting for Km^(r) and counterselecting against sacB,strains in which the plasmid is lost and the chromosomal hbp isexchanged for its mutated allele can be isolated (see FIG. 13A). Toidentify a strain that had undergone allelic exchange, Southernhybridization analysis on a Km^(r), sucrose-resistant clone which hadlost its resistance to chloramphenicol was performed (see FIG. 13B). Tosimultaneously confirm that the strain contained a Km^(r) insertionwhile lacking other vector sequences, pBOC22 was used as the probe. Aspredicted, this strain, which was designated NU226, had a 2.5-kbhbp-containing NdeI fragment in place of a 0.8-kb hybridizing NdeIfragment (compare lanes c and d). Southern hybridization analysis ofgenomic DNAs digested with HincII, HindIII, PstI, and PvuI furtherconfirmed that NU226 underwent allelic exchange (FIG. 13B, lanes a andb; and data not shown).

To ascertain whether hbp is associated with hemin binding in L.pneumophila, NU226 and 130b were grown on YP-minus-Fe-plus-hemin agarand then compared for their abilities to remove hemin from solution. Themutant strain displayed a 42% reduction in hemin binding, indicatingthat although hbp is not essential, it does enhance the acquisition ofhemin by Legionella organisms.

To determine whether hbp is required for intracellular infection ofmacrophages by L. pneumophila, we assessed the relative ability of NU226to infect U937 cells. Regardless of whether the inocula were preparedfrom YP-minus-Fe-plus hemin or BCYE cultures, the mutant exhibited a 50%infective dose that was comparable to that of the wild-type (data notshown). Furthermore, the intracellular growth pattern of NU226 wasnearly identical to that of strain 130b (FIG. 14). To explore thepossibility that hbp is important for growth within iron-depleted hostcells, the infections of U937 cells were repeated using U937 cells whichhad been treated with DFX. However, in two experiments, comparablenumbers of mutant and wild-type bacteria were recovered after 72 h ofincubation (data not shown). Finally, NU226 was not impaired in itsability to infect a protozoan host, the amoeba H. vermiformis (data notshown). Taken together, these results indicate that, although hbp is apromoter of hemin binding, it is not required for intracellularinfection by L. pneumophila.

4. Distribution of hbp Among L. pneumophila Strains and Other LegionellaSpecies. The L. pneumophila species consists of 14 serogroups, and theLegionella genus includes 39 species. Dennis, et al., Int. J. Syst.Bacteriol. 43:329-337 (1993). To determine whether hbp is conservedamong L. pneumophila strains and Legionella species, Southernhybridization analysis was performed using an hbp-specific probe. Underhigh-stringency conditions, the probe hybridized with DNAs from all L.pneumophila strains tested, suggesting that hbp is well conserved withinthe L. pneumophila species (see FIG. 15A). In contrast, hbp was largelyabsent from other Legionella species. Not only did the probe fail tohybridize with DNAs from these species under high stringency (data notshown); in most cases, it did not even hybridize underreduced-stringency conditions (see FIG. 15B). Curiously, L. moravica, anenvironmental isolate, and L. hackeliae, a clinical isolate, were theonly species that reacted with the hbp probe. Furthermore, thesehybridizations were quite weak compared with those of L. pneumophila(FIG. 15B, lanes a, e, and g). In a control Southern blot, DNAs from allof the Legionella spp. hybridized, as expected, with a mip probe underlow-stringency conditions (data not shown). Cianciotto, et al., Infect.Immun. 58:2912-2918 (1990). Taken together, these experiments indicatethat although hbp is not required for intracellular infection, it isnearly exclusive to the most pathogenic of Legionella species.

C. Discussion

With the results presented here, L. pneumophila now shares with a numberof other pathogens the ability to bind and utilize hemin as an ironsource. Furthermore, the capacity of hemin to replace ferric iron in aCDM indicates that it can be the sole source of iron. This experiencewith hemin also suggests that L. pneumophila can bind and use a varietyof heme-containing compounds. Feeley, et al., J. Clin. Microbiol.8:320-325 (1978); Otto, et al., Crit. Rev. Microbiol. 18:217-233 (1992);Wooldridge and Williams, FEMS Microbiol. Rev. 12:325-348 (1993). Sincecultures supplemented with hemin did not achieve the same degree ofgrowth as those supplemented with ferric PP_(i), ferric salts shouldnevertheless remain the preferred iron component of Legionella media.The level of hemin binding associated with the virulent strain 130b(i.e., 50 to 60% of added hemin) is quite comparable to those of otherpathogens. Daskaleros and Payne, Infect. Immun. 55:1393-1398 (1987);Deneer and Potter, Infect. Immun. 57:798-804 (1989); Genco, et al.,Infect. Immun. 62:2885-2892 (1994); Hanson and Hansen, Mol. Microbiol.5:267-278 (1991). In other systems, hemin binding is followed by theinternalization of the heme moiety, with the extraction of ironoccurring within an intracellular compartment. Daskaleros, et al.,Infect. Immun. 59:2706-2711 (1991); Mills and Payne, J. Bacteriol.177:3004-3009 (1995); Stojiljkovic and Hantke, Mol. Microbiol.13:719-732 (1994). However, a recent report on N. gonorrhoeae indicatesthat, in some cases, iron is extracted from hemin at the cell surface.Desai P. J., et al., Infect. Immun. 63:4634-4641 (1995). Clearly,transport studies with radiolabeled hemin are needed to ascertain whichheme iron assimilation pathway is operative in the legionellae. However,the inability of hemin to restore growth to strain 130b culturescontaining the ferric iron chelator DFX (data not shown) suggests thatL. pneumophila is akin to N. gonorrhoeae. The observation thatlegionellae harvested from YP-minus-Fe-plus-hemin media exhibited abinding activity greater than those harvested from BCYE media warrantsadditional comment. Two explanations for this observation can beoffered. First, it is possible that in Legionella, as in other microbes,the presence of hemin (in the YP medium) induces the expression of heminacquisition functions. Carman, et al., Infect. Immun. 58:4016-4019(1990); Genco, et al., Infect. Immun. 62:2885-2892 (1994). Alternately,the presence of a high level of iron (in the BCYE medium) represseshemin-binding activity. Henderson and Payne, J. Bacteriol. 176:3269-3277(1994); Perry, Trends Microbiol. 1:142-147 (1993); Worst, et al.,Infect. Immun. 63:4161-4165 (1995). The latter possibility is supportedby the placement of an iron box-like sequence upstream of hbp.

In L. pneumophila, as in other microbes, hemin binding and utilizationundoubtedly require the action of numerous genes. For five reasons, itis strongly suspected that the newly identified hbp is one of thosegenes. First, when cloned into E. coli, it promoted hemin binding bothon plates and in liquid. Second, the extent of that binding wascomparable to what is observed with genes cloned from other bacteria.Hanson and Hansen, Mol. Microbiol. 5:267-278 (1991). Third, the clonedhbp also conferred Congo red binding, which is a trait often associatedwith hemin binding. Daskaleros and Payne, Infect. Immun. 55:1393-1398(1987); Deneer and Potter, Infect. Immun. 57:798-804 (1989); Genco, etal., Infect. Immun. 62:2885-2892 (1994); Kay, et al., J. Bacteriol.164:1332-1336 (1985); Stugard, et al., Infect. Immun. 57:3534-3539(1989). Fourth, the predicted product of hbp appears to be secreted, acharacteristic that is common to proteins which are specificallyassociated with hemin acquisition. Bramanti and Holt, J. Bacteriol.175:7413-7420 (1993); Cope, et al., J. Bacteriol. 177:2644-2653 (1995);Elkins, et al., Infect. Immun. 63:2194-2200 (1995); Hanson and Hansen,Mol. Microbiol. 5:267-278 (1991); Henderson and Payne, J. Bacteriol.176:3269-3277 (1994); Lewis and Dyer, J. Bacteriol. 177:1299-1306(1995); Mills and Payne, J. Bacteriol. 177:3004-3009 (1995);Stojiljkovic and Hantke, Mol. Microbiol. 13:719-732 (1994). Fifth, andmost importantly, an L. pneumophila strain containing a DNA insertionwithin the monocistronic hbp displayed a 43% reduction in hemin binding.It is not clear how hbp (Hbp) potentiates hemin acquisition in L.pneumophila or E. coli. In one scenario, Hbp is the surface protein thatdirectly binds hemin. In a second scenario, it facilitates theexpression or activity of another, possibly conserved, component of thegram-negative cell wall. The fact that the Legionella hbp mutant stillexhibited some hemin binding would tend to support the second scenario.The absence of a second membrane-spanning domain within Hbp is mostcompatible with a periplasmic rather than outer membrane location forthe protein. A hemin-binding protein of H. influenze which wasidentified by screening a genomic library on hemin plates is believed tobe located, at least in part, within the periplasm. Hanson and Hansen,Mol. Microbiol. 5:267-278 (1991).

In three different assays, the L. pneumophila hbp mutant was notimpaired in its ability to infect eukaryotic hosts, clearly indicatingthat hbp is not required for intracellular infection. This resultdiminishes significantly but does not eliminate the possibility that hbpencodes a virulence factor; i.e., only experimental animal infectionscan ascertain whether hbp promotes (extracellular) survival and/orgrowth in vivo. For two reasons, this result also does not necessarilymean that hemin acquisition and utilization are not critical forintracellular L. pneumophila growth. On the one hand, the residual heminbinding displayed by the mutant may suffice inside host cells. On theother hand, intracellular legionellae might express other genes whichcan restore hemin binding to its full capacity.

Southern hybridization analysis indicated that hbp sequences are nearlylimited to the L. pneumophila species. This type of gene distribution israther peculiar. When assessed, all previously identified L. pneumophilagenes, including flaA, fur, htp, lly, mip, ompS, and ppLa (pal), havehad homologs in all or virtually all other Legionella species. Bender,et al., Infect. Immun. 59:3333-3336 (1991); Cianciotto, et al., Infect.Immun. 58:2912-2918 (1990); Heuner, et al., Infect. Immun. 63:2499-2507(1995); Hickey and Cianciotto, Gene 143:117-121 (1994); Hoffman, et al.,Infect. Immn. 57:1731-1739 (1989); Hoffman, et al., J. Bacteriol.174:914-920 (1992); Ott, et al., Microb. Pathog. 11:357-365 (1991).Although the biological significance of this observation is unclear, thelimited distribution of hbp among the legionellae warrants its use as adiagnostic reagent. Currently, mip primers are used for the PCRdetection of L. pneumophila within water and clinical samples. Atlas, etal., Appl. Environ. Microbiol. 61:1208-1213 (1995); Bej, et al., Appl.Environ. Microbiol. 57:597-600 (1991); Jaulhac, et al., J. Clin.Microbiol. 30:920-924 (1992); King, et al., Infect. Immun. 59:758-763(1991); Lindsay, et al., J. Clin. Microbiol. 32:3068-3069 (1994);Oshiro, et al., Can. J. Microbiol. 40:495-499 (1994). However, inseveral instances, these primers amplify DNAs (i.e., mip-like genes)from other Legionella species. Atlas, et al., Appl. Environ. Microbiol.61:1208-1213 (1995); Cianciotto, et al., Infect. Immun. 58:2912-2918(1990); Jaulhac, et al., J. Clin. Microbiol. 30:920-924 (1992); Oshiro,et al., Can. J. Microbiol. 40:495-499 (1994). Thus, hbp primers shouldenhance the specificity of L. pneumophila PCR detection protocols.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 47    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Primer"A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #                23TAAA ACG    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Primer"A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #                 22CAC AG    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Primer"A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #21                TTTA C    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1980 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - ATTTAACTCA GAATACAGAA ACTTTTTATC CAAAATCAAA CTCTATTGCC AA - #TTAATAAT      60    - ATTTTATTTG CCCCACCTCT TGCAAATGAG AATGATTATC ATTTATATTT AA - #TTTATAAC     120    - AAAATAATCC TTCAGGAGAT AATA ATG GCC CTG GCG TAC G - #GT AAT TTT CAT     171    #Gly Asn Phe Hisa Leu Ala Tyr    #        5  1    - GAA CTC AGC CAT CAA TTA CGC TTT TTA CTA TT - #T GAA ATT GGC ATA GGA     219    Glu Leu Ser His Gln Leu Arg Phe Leu Leu Ph - #e Glu Ile Gly Ile Gly    # 25    - CTA CCA CAA AAT AGT GTG GAT TAT TTT ATT AC - #C TTA GCT CAT AAA AAT     267    Leu Pro Gln Asn Ser Val Asp Tyr Phe Ile Th - #r Leu Ala His Lys Asn    #                 40    - ACC CTG AAG CGT TTA CAG CAT GCC TCC ATT AA - #G GAA GGA TTA ATT CAA     315    Thr Leu Lys Arg Leu Gln His Ala Ser Ile Ly - #s Glu Gly Leu Ile Gln    #             55    - TCA GCC ATT GCA AGT CAC CAT ATC CAT GAT TT - #C ATT GAC CAA TTG CAG     363    Ser Ala Ile Ala Ser His His Ile His Asp Ph - #e Ile Asp Gln Leu Gln    #         70    - ATA AAA CTG AAA AAT TCA ATG CCG GAA AGT AA - #G TTT TTT CAA TGG CGA     411    Ile Lys Leu Lys Asn Ser Met Pro Glu Ser Ly - #s Phe Phe Gln Trp Arg    #     85    - AAA ATC AGG GAA GCA TTA GAT GAA TCG ATT GC - #C AAT GAG GCT TTG GCT     459    Lys Ile Arg Glu Ala Leu Asp Glu Ser Ile Al - #a Asn Glu Ala Leu Ala    #105    - TAC GCC TAC AGG CAA AAC TGG AAC ACC CAA TT - #A AGA AAT GAA GCC ATG     507    Tyr Ala Tyr Arg Gln Asn Trp Asn Thr Gln Le - #u Arg Asn Glu Ala Met    #               120    - CAC TAC AAG AGT CTG TGG ACA TGG ATA AAT AA - #T GAA CTA TCT CCG TAT     555    His Tyr Lys Ser Leu Trp Thr Trp Ile Asn As - #n Glu Leu Ser Pro Tyr    #           135    - CAA ACG TTA TTA TTT CTG GAA CAA TGG GGC AG - #T TTG AGG CAT CCC TAT     603    Gln Thr Leu Leu Phe Leu Glu Gln Trp Gly Se - #r Leu Arg His Pro Tyr    #       150    - CAC CCA GCA TTC AGC GCA AAA ACA GGG TTT AC - #G CGA AGA GAA GTA CTC     651    His Pro Ala Phe Ser Ala Lys Thr Gly Phe Th - #r Arg Arg Glu Val Leu    #   165    - CAA AAC TCT CCC GAA TTC CAG GCC AAA GTC AG - #T GTA CAT TGG TGT GCA     699    Gln Asn Ser Pro Glu Phe Gln Ala Lys Val Se - #r Val His Trp Cys Ala    170                 1 - #75                 1 - #80                 1 -    #85    - TTA AAT AAA ACA AAA ATT CAG TCA ATA AGC CC - #A AAA ATT GAT TAT GCC     747    Leu Asn Lys Thr Lys Ile Gln Ser Ile Ser Pr - #o Lys Ile Asp Tyr Ala    #               200    - AAC CAA ATT TCT CAA GAA TTT CCC AAA GAA TA - #T TTT TAT TGG CGT GAA     795    Asn Gln Ile Ser Gln Glu Phe Pro Lys Glu Ty - #r Phe Tyr Trp Arg Glu    #           215    - AAA TTG TTA TTT AGC CAC ATC AAC CCT GAT GA - #T TAT TAT CCA ATT CCT     843    Lys Leu Leu Phe Ser His Ile Asn Pro Asp As - #p Tyr Tyr Pro Ile Pro    #       230    - GTT CAC CCT TGG CAG TGG AGG AAT CAA TTA CA - #A ATG GCG TTT GCA TCT     891    Val His Pro Trp Gln Trp Arg Asn Gln Leu Gl - #n Met Ala Phe Ala Ser    #   245    - TTA ATT GAT AAT AAA TCC CTC ATC TTG TTA CC - #T CAT CAC CAA ACA CTA     939    Leu Ile Asp Asn Lys Ser Leu Ile Leu Leu Pr - #o His His Gln Thr Leu    250                 2 - #55                 2 - #60                 2 -    #65    - ATA CCT TCT TTA TCA CCT GAT ATT ATG ATG CC - #A ACA CAA TCC ACT CAA     987    Ile Pro Ser Leu Ser Pro Asp Ile Met Met Pr - #o Thr Gln Ser Thr Gln    #               280    - TGT ACA CTT AAA CTG GCT ACC ACT TTA AGC AC - #C TCA ATG GCT GGA AAA    1035    Cys Thr Leu Lys Leu Ala Thr Thr Leu Ser Th - #r Ser Met Ala Gly Lys    #           295    - CTT GAT AAT TCT AAT GAT ATG GTA TTG CTT AC - #C AGA TGG ATC GAT TCC    1083    Leu Asp Asn Ser Asn Asp Met Val Leu Leu Th - #r Arg Trp Ile Asp Ser    #       310    - CTG TTA GCA AAA ACA AAC TAT TAC CAA AAT AC - #C TTG TTT ATA TGT AAA    1131    Leu Leu Ala Lys Thr Asn Tyr Tyr Gln Asn Th - #r Leu Phe Ile Cys Lys    #   325    - AAC CTG GAG AGC ATG AGC GCC TAT GAT CAA AC - #T CTC TCT GAA TGC AAT    1179    Asn Leu Glu Ser Met Ser Ala Tyr Asp Gln Th - #r Leu Ser Glu Cys Asn    330                 3 - #35                 3 - #40                 3 -    #45    - CGA GTA AAA TTA TTG TTT GGC TTA TAT CAA AA - #C CCA CTC CAC AAA ATA    1227    Arg Val Lys Leu Leu Phe Gly Leu Tyr Gln As - #n Pro Leu His Lys Ile    #               360    - AGA CAG GAT CAA AGA GCA GTT CCA TTA CCT GC - #C CTA TTA ACT GAT TCC    1275    Arg Gln Asp Gln Arg Ala Val Pro Leu Pro Al - #a Leu Leu Thr Asp Ser    #           375    - CCT TGT AGC AAT ACA CCA TTA CTC ATT GAA AT - #T ATT AAA GCT AGC GGC    1323    Pro Cys Ser Asn Thr Pro Leu Leu Ile Glu Il - #e Ile Lys Ala Ser Gly    #       390    - CTG CAC CCA ACG ACT TAT TTC ACT GAA TAT TG - #T TAT AAG ATG TTA TTT    1371    Leu His Pro Thr Thr Tyr Phe Thr Glu Tyr Cy - #s Tyr Lys Met Leu Phe    #   405    - GGA CAA TTG CAT CTA TTG CTA AAA TAT GGA TT - #A GCA CTA GAA GTG GAG    1419    Gly Gln Leu His Leu Leu Leu Lys Tyr Gly Le - #u Ala Leu Glu Val Glu    410                 4 - #15                 4 - #20                 4 -    #25    - CAA CAC AAT ATT TTA GTC ATC TTC GAT GAC AA - #T AAA CCT CAG GGG ATA    1467    Gln His Asn Ile Leu Val Ile Phe Asp Asp As - #n Lys Pro Gln Gly Ile    #               440    - ATT ATA AAA GAG CCA AAC AAC CTT AAG CTA TG - #C AAT CAT GAA CTG TTT    1515    Ile Ile Lys Glu Pro Asn Asn Leu Lys Leu Cy - #s Asn His Glu Leu Phe    #           455    - AAA AAC GTT CAA AAA CCC AAC GCT CCA GAC TC - #T TTA TCC ATC TAT ACA    1563    Lys Asn Val Gln Lys Pro Asn Ala Pro Asp Se - #r Leu Ser Ile Tyr Thr    #       470    - AAA GAT CTT AAT CAG GTT AGA ACC CTT TTC AT - #C CAG GGA ACA TTA AAA    1611    Lys Asp Leu Asn Gln Val Arg Thr Leu Phe Il - #e Gln Gly Thr Leu Lys    #   485    - AAT CAT CTA CAT CAC TTG ATT GGC TGT TTA CG - #T AAT GAG TAT CAG ATT    1659    Asn His Leu His His Leu Ile Gly Cys Leu Ar - #g Asn Glu Tyr Gln Ile    490                 4 - #95                 5 - #00                 5 -    #05    - CCT TCA AGA ACC TTA TGG GGA TTA GCT CGC CA - #A GTC ATG CAA ACT GTA    1707    Pro Ser Arg Thr Leu Trp Gly Leu Ala Arg Gl - #n Val Met Gln Thr Val    #               520    - TTT AAA GAC TTA TCC AAA GAC ATT GAT CCG CG - #T ATT CTA AGT TGG CAA    1755    Phe Lys Asp Leu Ser Lys Asp Ile Asp Pro Ar - #g Ile Leu Ser Trp Gln    #           535    - CAA CAT CTA TTG CTT CAT GAT AAC TGG GAG CA - #T CAA CCT GAA TTG TTA    1803    Gln His Leu Leu Leu His Asp Asn Trp Glu Hi - #s Gln Pro Glu Leu Leu    #       550    - TTA AGT CTG CAT TCC AAA ATC AAT CGA AAT AT - #T ACA ATA AAG GAA TAC    1851    Leu Ser Leu His Ser Lys Ile Asn Arg Asn Il - #e Thr Ile Lys Glu Tyr    #   565    #AAAACCCCAT          1900 T AAGCTCTACT GGACTTACGA    Asn Pro Leu Ser Glu Ile    570                 5 - #75    - GCGCTCTTGT TCCAATACTA AAATATGAGA ACTTCTCGAG GCCGGGATGT GG - #ATACTATG    1960    #                 198 - #0    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 575 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Met Ala Leu Ala Tyr Gly Asn Phe His Glu Le - #u Ser His Gln Leu Arg    #                 15    - Phe Leu Leu Phe Glu Ile Gly Ile Gly Leu Pr - #o Gln Asn Ser Val Asp    #             30    - Tyr Phe Ile Thr Leu Ala His Lys Asn Thr Le - #u Lys Arg Leu Gln His    #         45    - Ala Ser Ile Lys Glu Gly Leu Ile Gln Ser Al - #a Ile Ala Ser His His    #     60    - Ile His Asp Phe Ile Asp Gln Leu Gln Ile Ly - #s Leu Lys Asn Ser Met    # 80    - Pro Glu Ser Lys Phe Phe Gln Trp Arg Lys Il - #e Arg Glu Ala Leu Asp    #                 95    - Glu Ser Ile Ala Asn Glu Ala Leu Ala Tyr Al - #a Tyr Arg Gln Asn Trp    #           110    - Asn Thr Gln Leu Arg Asn Glu Ala Met His Ty - #r Lys Ser Leu Trp Thr    #       125    - Trp Ile Asn Asn Glu Leu Ser Pro Tyr Gln Th - #r Leu Leu Phe Leu Glu    #   140    - Gln Trp Gly Ser Leu Arg His Pro Tyr His Pr - #o Ala Phe Ser Ala Lys    145                 1 - #50                 1 - #55                 1 -    #60    - Thr Gly Phe Thr Arg Arg Glu Val Leu Gln As - #n Ser Pro Glu Phe Gln    #               175    - Ala Lys Val Ser Val His Trp Cys Ala Leu As - #n Lys Thr Lys Ile Gln    #           190    - Ser Ile Ser Pro Lys Ile Asp Tyr Ala Asn Gl - #n Ile Ser Gln Glu Phe    #       205    - Pro Lys Glu Tyr Phe Tyr Trp Arg Glu Lys Le - #u Leu Phe Ser His Ile    #   220    - Asn Pro Asp Asp Tyr Tyr Pro Ile Pro Val Hi - #s Pro Trp Gln Trp Arg    225                 2 - #30                 2 - #35                 2 -    #40    - Asn Gln Leu Gln Met Ala Phe Ala Ser Leu Il - #e Asp Asn Lys Ser Leu    #               255    - Ile Leu Leu Pro His His Gln Thr Leu Ile Pr - #o Ser Leu Ser Pro Asp    #           270    - Ile Met Met Pro Thr Gln Ser Thr Gln Cys Th - #r Leu Lys Leu Ala Thr    #       285    - Thr Leu Ser Thr Ser Met Ala Gly Lys Leu As - #p Asn Ser Asn Asp Met    #   300    - Val Leu Leu Thr Arg Trp Ile Asp Ser Leu Le - #u Ala Lys Thr Asn Tyr    305                 3 - #10                 3 - #15                 3 -    #20    - Tyr Gln Asn Thr Leu Phe Ile Cys Lys Asn Le - #u Glu Ser Met Ser Ala    #               335    - Tyr Asp Gln Thr Leu Ser Glu Cys Asn Arg Va - #l Lys Leu Leu Phe Gly    #           350    - Leu Tyr Gln Asn Pro Leu His Lys Ile Arg Gl - #n Asp Gln Arg Ala Val    #       365    - Pro Leu Pro Ala Leu Leu Thr Asp Ser Pro Cy - #s Ser Asn Thr Pro Leu    #   380    - Leu Ile Glu Ile Ile Lys Ala Ser Gly Leu Hi - #s Pro Thr Thr Tyr Phe    385                 3 - #90                 3 - #95                 4 -    #00    - Thr Glu Tyr Cys Tyr Lys Met Leu Phe Gly Gl - #n Leu His Leu Leu Leu    #               415    - Lys Tyr Gly Leu Ala Leu Glu Val Glu Gln Hi - #s Asn Ile Leu Val Ile    #           430    - Phe Asp Asp Asn Lys Pro Gln Gly Ile Ile Il - #e Lys Glu Pro Asn Asn    #       445    - Leu Lys Leu Cys Asn His Glu Leu Phe Lys As - #n Val Gln Lys Pro Asn    #   460    - Ala Pro Asp Ser Leu Ser Ile Tyr Thr Lys As - #p Leu Asn Gln Val Arg    465                 4 - #70                 4 - #75                 4 -    #80    - Thr Leu Phe Ile Gln Gly Thr Leu Lys Asn Hi - #s Leu His His Leu Ile    #               495    - Gly Cys Leu Arg Asn Glu Tyr Gln Ile Pro Se - #r Arg Thr Leu Trp Gly    #           510    - Leu Ala Arg Gln Val Met Gln Thr Val Phe Ly - #s Asp Leu Ser Lys Asp    #       525    - Ile Asp Pro Arg Ile Leu Ser Trp Gln Gln Hi - #s Leu Leu Leu His Asp    #   540    - Asn Trp Glu His Gln Pro Glu Leu Leu Leu Se - #r Leu His Ser Lys Ile    545                 5 - #50                 5 - #55                 5 -    #60    - Asn Arg Asn Ile Thr Ile Lys Glu Tyr Asn Pr - #o Leu Ser Glu Ile    #               575    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 44 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Met Ile Leu Pro Ser Glu Lys Ser Ala Thr As - #p Val Ala Ala Gln Cys    #                15    - Phe Leu Asn Ala Leu Ile Arg Glu Thr Lys As - #p Trp Gln Leu Ala Glu    #            30    - Tyr Pro Pro Asp Glu Leu Ile Ile Pro Leu As - #p Glu    #        40    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Met Ala Leu Ala Tyr Gly Asn Phe His Glu Le - #u Ser His Gln Leu Arg    #                15    - Phe Leu Leu Phe Glu Ile Gly Ile Gly Leu Pr - #o Gln Asn Ser Val Asp    #            30    - Tyr Phe Ile Thr Leu Ala His Lys Asn Thr Le - #u Lys Arg Leu Gln    #        45    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 40 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Met Asn His Lys Asp Trp Asp Leu Val Asn Ar - #g Arg Leu Val Ala Lys    #                15    - Met Leu Ser Glu Leu Glu Tyr Glu Gln Val Ph - #e His Ala Glu Ser Gln    #            30    - Gly Asp Asp Arg Tyr Cys Ile Asn    #        40    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Gln Lys Ser Leu His Phe Arg Val Ala Tyr Ph - #e Ser Pro Thr Gln His    #                15    - His Arg Phe Ala Phe Pro Ala His Leu Val Th - #r Ala Ser Gly Ser Tyr    #            30    - Pro Val Asp Phe Thr Thr Leu Ser Arg Leu Il - #e Ile Asp Lys Leu Arg    #        45    - His Gln        50    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - His Ala Ser Ile Lys Glu Gly Leu Ile Gln Se - #r Ala Ile Ala Ser His    #                15    - His Ile His Asp Phe Ile Asp Gln Leu Gln Il - #e Lys Leu Lys Asn Ser    #            30    - Met Pro Glu Ser Lys Phe Phe Gln Trp Arg Ly - #s Ile Arg Glu Ala Leu    #        45    - Asp Glu        50    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - Leu Pro Gly Ala Gln Trp Arg Phe Ile Ala Gl - #u Arg Gly Ile Trp Gly    #                15    - Trp Leu Trp Ile Asp Ala Gln Thr Leu Arg Cy - #s Ala Asp Glu Pro Val    #            30    - Leu Ala Gln Thr Leu Leu Met Gln Leu Lys Gl - #n Val Leu Ser Met Ser    #        45    - Asp Ala        50    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - Leu Phe Leu Pro Val Pro Leu Cys Glu Thr Ph - #e His Gln Arg Val Leu    #                15    - Glu Ser Tyr Ala His Thr Gln Gln Thr Ile As - #p Ala Arg His Asp Trp    #            30    - Ala Ile Leu Arg Glu Lys Ala Leu Asn Phe Gl - #y Glu Ala Glu Gln    #        45    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    - Ser Ile Ala Asn Glu Ala Leu Ala Tyr Ala Ty - #r Arg Gln Asn Trp Asn    #                15    - Thr Gln Leu Arg Asn Glu Ala Met His Tyr Ly - #s Ser Leu Trp Thr Trp    #            30    - Ile Asn Asn Glu Leu Ser Pro Tyr Gln Thr Le - #u Leu Phe Leu Glu Gln    #        45    - Trp Gly        50    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 44 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    - Thr Val Ala Glu His Met Gln Asp Leu Tyr Al - #a Thr Leu Leu Gly Asp    #                15    - Leu Gln Leu Leu Lys Ala Arg Arg Gly Leu Se - #r Ala Ser Asp Leu Ile    #            30    - Asn Leu Asn Ala Asp Arg Leu Gln Cys Leu Le - #u Ser    #        40    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 44 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    - Ala Leu Leu Thr Gly His Ala Phe His Pro Al - #a Pro Lys Ser His Glu    #                15    - Pro Phe Asn Arg Gln Glu Ala Glu Arg Tyr Le - #u Pro Asp Met Ala Pro    #            30    - His Phe Pro Leu Arg Trp Phe Ser Val Asp Ly - #s Thr    #        40    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    - Ser Leu Arg His Pro Tyr His Pro Ala Phe Se - #r Ala Lys Thr Gly Phe    #                15    - Thr Arg Arg Glu Val Leu Gln Asn Ser Pro Gl - #u Phe Gln Ala Lys Val    #            30    - Ser Val His Trp Cys Ala Leu Asn Lys Thr    #        40    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 46 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - Gly His Pro Lys Phe Val Phe Asn Lys Gly Ar - #g Arg Gly Trp Gly Lys    #                15    - Glu Ala Leu Glu Arg Tyr Ala Pro Glu Tyr Al - #a Asn Thr Phe Arg Leu    #            30    - His Trp Leu Ala Val Lys Arg Glu His Met Il - #e Trp Arg Cys    #        45    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 44 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - Gln Ile Ala Gly Glu Ser Leu His Leu Asn Le - #u Gln Gln Arg Leu Thr    #                15    - Arg Phe Ala Ala Glu Asn Ala Pro Gln Leu Le - #u Asn Glu Leu Ser Asp    #            30    - Asn Gln Trp Leu Phe Pro Leu Arg Pro Trp Gl - #n Gly    #        40    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    - Lys Ile Gln Ser Ile Ser Pro Lys Ile Asp Ty - #r Ala Asn Gln Ile Ser    #                15    - Gln Glu Phe Pro Lys Glu Tyr Phe Tyr Trp Ar - #g Glu Lys Leu Leu Phe    #            30    - Ser His Ile Asn Pro Asp Asp Tyr Tyr Pro Il - #e Pro Val His Pro Trp    #        45    - Gln Trp        50    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    - Asp Asn Glu Met Asp Ile His Gln Leu Leu Th - #r Ala Ala Met Asp Pro    #                15    - Gln Glu Phe Ala Arg Phe Ser Gln Val Trp Gl - #n Glu Asn Gly Leu Asp    #            30    - His Asn Trp Leu Pro Leu Pro Val His Pro Tr - #p Gln Trp    #        45    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 48 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    - Glu Tyr Leu Phe Gln Gln Val Trp Cys Gln Al - #a Leu Phe Ala Lys Gly    #                15    - Leu Ile Arg Asp Leu Gly Glu Ala Gly Thr Se - #r Trp Leu Pro Thr Thr    #            30    - Ser Ser Arg Ser Leu Tyr Cys Ala Thr Ser Ar - #g Asp Met Ile Lys Phe    #        45    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    - Arg Asn Gln Leu Gln Met Ala Phe Ala Ser Le - #u Ile Asp Asn Lys Ser    #                15    - Leu Ile Leu Leu Pro His His Gln Thr Leu Il - #e Pro Ser Leu Ser Pro    #            30    - Asp Ile Met Met Pro Thr Gln Ser Thr Gln Cy - #s Thr Leu    #        45    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    - Gln Glu Lys Ile Ala Thr Asp Phe Ile Ala As - #p Phe Gly Glu Gly Arg    #                15    - Met Val Ser Leu Gly Glu Phe Gly Asp Gln Tr - #p Leu Ala Gln Gln Ser    #            30    - Leu Arg Thr Leu Thr Asn Ala Ser Arg Arg Gl - #y Gly Leu Asp Ile    #        45    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    - Ser Leu Ser Val Arg Leu Thr Asn Ser Val Ar - #g Thr Leu Ser Val Lys    #                15    - Glu Val Glu Arg Gly Met Arg Leu Ala Arg Le - #u Ala Gln Thr Asp Gly    #            30    - Trp Gln Met Leu Gln Ala Arg Phe Pro Thr Ph - #e Arg Val Met Gln    #        45    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    - Lys Leu Ala Thr Thr Leu Ser Thr Ser Met Al - #a Gly Lys Leu Asp Asn    #                15    - Ser Asn Asp Met Val Leu Leu Thr Arg Trp Il - #e Asp Ser Leu Leu Ala    #            30    - Lys Thr Asn Tyr Tyr Gln Asn Thr Leu Phe Il - #e Cys Lys Asn Leu    #        45    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 48 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    - Lys Leu Pro Leu Thr Ile Tyr Asn Thr Ser Cy - #s Tyr Arg Gly Ile Pro    #                15    - Gly Arg Tyr Ile Ala Ala Gly Pro Leu Ala Se - #r Arg Trp Leu Gln Gln    #            30    - Val Phe Ala Thr Asp Ala Thr Leu Val Gln Se - #r Gly Ala Val Ile Leu    #        45    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    - Glu Asp Asp Trp Thr Gly Leu Arg Asp Leu As - #n Gly Asn Ile Met Gln    #                15    - Glu Ser Leu Phe Ser Pro Ala Trp Lys Thr Le - #u Leu Leu Glu Gln Pro    #            30    - Gln Ser Gln Thr Asn Val Leu Val Ser Leu    #        40    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    - Glu Ser Met Ser Ala Tyr Asp Gln Thr Leu Se - #r Glu Cys Asn Arg Val    #                15    - Lys Leu Leu Phe Gly Leu Tyr Gln Asn Pro Le - #u His Lys Ile Arg Gln    #            30    - Asp Gln Arg Ala Val Pro Leu Pro Ala Leu    #        40    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    - Gly Glu Pro Ala Ala Gly Tyr Val Ser His Gl - #u Gly Tyr Ala Ala Leu    #                15    - Ala Arg Ala Pro Tyr Arg Tyr Gln Glu Met Le - #u Gly Val Ile Trp Arg    #            30    - Glu Asn Pro Cys Arg Trp Leu Lys Pro Asp Gl - #u Ser Pro Phe Leu Met    #        45    - Ala Thr        50    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    - Thr Gln Ala Gly Pro His Gly Gly Asp Ser Le - #u Leu Val Ser Ala Val    #                15    - Lys Arg Leu Ser Asp Arg Leu Gly Ile Thr Va - #l Gln Gln Ala Ala His    #            30    - Ala Trp Val Asp Ala Tyr Cys Gln Gln Val Le - #u Lys Pro Leu Phe Thr    #        45    - Ala Glu        50    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    - Leu Thr Asp Ser Pro Cys Ser Asn Thr Pro Le - #u Leu Ile Glu Ile Ile    #                15    - Lys Ala Ser Gly Leu His Pro Thr Thr Tyr Ph - #e Thr Glu Tyr Cys Tyr    #            30    - Lys Met Leu Phe Gly Gln Leu His Leu Leu    #        40    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    - Leu Met Glu Trp Asp Glu Asn Asn Gln Pro Le - #u Ala Gly Ala Tyr Ile    #                15    - Asp Arg Ser Gly Leu Asp Ala Glu Thr Trp Le - #u Thr Gln Leu Phe Arg    #            30    - Val Val Val Val Pro Leu Tyr His Leu Leu    #        40    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 49 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    - Ala Asp Tyr Gly Leu Val Leu Leu Ala His Gl - #n Gln Asn Ile Leu Val    #                15    - Gln Met Leu Gly Asp Leu Pro Val Gly Phe Il - #e Tyr Arg Asp Cys Gln    #            30    - Gly Ser Ala Phe Met Pro His Ala Thr Glu Tr - #p Leu Asp Thr Ile Asp    #        45    - Glu    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    - Leu Lys Tyr Gly Leu Ala Leu Glu Val Glu Gl - #n His Asn Ile Leu Val    #                15    - Ile Phe Asp Asp Asn Lys Pro Gln Gly Ile Il - #e Ile Lys Glu Pro Asn    #            30    - Asn Leu Lys Leu Cys Asn His Glu Leu Phe Ly - #s Asn Val Gln Lys    #        45    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    - Cys Arg Tyr Gly Val Ala Leu Ile Ala His Gl - #y Gln Asn Ile Thr Leu    #                15    - Ala Met Lys Glu Gly Val Pro Gln Arg Val Le - #u Leu Lys Asp Phe Gln    #            30    - Gly Asp Met Arg Leu Val Lys Glu Glu Phe    #        40    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 47 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    - Ala Gln Ala Glu Asn Ile Phe Thr Arg Glu Gl - #n Leu Leu Arg Tyr Phe    #                15    - Pro Tyr Tyr Leu Leu Val Asn Ser Thr Phe Al - #a Val Thr Ala Ala Leu    #            30    - Gly Ala Ala Gly Leu Asp Ser Glu Ala Asn Le - #u Met Ala Arg Val    #        45    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 48 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    - Pro Asn Ala Pro Asp Ser Leu Ser Ile Tyr Th - #r Lys Asp Leu Asn Gln    #                15    - Val Arg Thr Leu Phe Ile Gln Gly Thr Leu Ly - #s Asn His Leu His His    #            30    - Leu Ile Gly Cys Leu Arg Asn Glu Tyr Gln Il - #e Pro Ser Arg Thr Leu    #        45    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    - Pro Glu Met Asp Ser Leu Pro Gln Glu Val Ar - #g Asp Val Thr Ser Arg    #                15    - Leu Ser Ala Asp Tyr Leu Ile His Asp Leu Gl - #n Thr Gly His Phe Val    #            30    - Thr Val Leu Arg Phe Ile Ser Pro Leu Met Va - #l Arg Leu    #        45    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    - Arg Thr Leu Leu Ala Glu Val Arg Asp Gln Va - #l Thr His Lys Thr Cys    #                15    - Leu Asn Tyr Val Leu Glu Ser Pro Tyr Trp As - #n Val Lys Gly Asn Phe    #            30    - Phe Cys Tyr Leu Asn Asp His Asn Glu Asn Th - #r Ile Val Asp Pro Ser    #        45    - Val Ile        50    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    - Trp Gly Leu Ala Arg Gln Val Met Gln Thr Va - #l Phe Lys Asp Leu Ser    #                15    - Lys Asp Ile Asp Pro Arg Ile Leu Ser Trp Gl - #n Gln His Leu Leu Leu    #            30    - His Asp Asn Trp Glu His Gln Pro Glu Leu Le - #u Leu Ser Leu His Ser    #        45    - Lys Ile        50    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 35 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    - Gly Val Pro Glu Arg Arg Phe Tyr Gln Leu Le - #u Ala Ala Val Leu Ser    #                15    - Asp Tyr Met Lys Lys His Pro Gln Met Ser Gl - #u Arg Phe Ala Leu Phe    #            30    - Ser Leu Phe            35    - (2) INFORMATION FOR SEQ ID NO:42:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    - Tyr Phe Asp Phe Ala Asn Pro Leu Gln Ala Gl - #n Glu Val    #                10    - (2) INFORMATION FOR SEQ ID NO:43:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    - Asn Arg Asn Ile Thr Ile Lys Glu Tyr Asn Pr - #o Leu Ser Glu Ile    #                15    - (2) INFORMATION FOR SEQ ID NO:44:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 11 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    - Arg Pro Gln Ile Ile Arg Val Val Leu Asn Pr - #o    #                10    - (2) INFORMATION FOR SEQ ID NO:45:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 653 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    - GAAAAATATC CTTATAAATA TGAATTAGCC ATTGCATATG GCAAAAGATA AT - #CTGAAACA      60    - ATTCCCGACA ACATCCTTTT AAAACGGTTG CAACTGAAAA TTCAACTTGT TA - #GTCTTTTG     120    #AAA ACC CAG      174GT AACAAGGAGA AGTAAC ATG ATG TTG    #    Met Met Leu Lys Thr Gln    #   5  1    - TTG ACT GCT TTT ATC GGT GCT GTA ATC TTG GC - #T GGC TCT TCT TTA GCA     222    Leu Thr Ala Phe Ile Gly Ala Val Ile Leu Al - #a Gly Ser Ser Leu Ala    #             20    - AAT CCA ATA AAA CCT GAG GTA TGC CCC AGT GT - #A CCC TCT ATT CAA TCG     270    Asn Pro Ile Lys Pro Glu Val Cys Pro Ser Va - #l Pro Ser Ile Gln Ser    #         35    - GAA GGA ATG TCC ATG TCT TCT GAA ATT TTG GA - #G GGC ATG TAC ATC ACC     318    Glu Gly Met Ser Met Ser Ser Glu Ile Leu Gl - #u Gly Met Tyr Ile Thr    #     50    - TAT AAT TTA AGT CAT TAC AAT ACC AGT TCA AG - #C TGG GTG TTT ATT GTA     366    Tyr Asn Leu Ser His Tyr Asn Thr Ser Ser Se - #r Trp Val Phe Ile Val    # 70    - GGG CCA ATC GCA GCT GAA AAT GAT GAT ATG GC - #A TTG GCA GAA AGC AAT     414    Gly Pro Ile Ala Ala Glu Asn Asp Asp Met Al - #a Leu Ala Glu Ser Asn    #                 85    - AAA TTA CTT TCA ACC ATG TCA GGG TCT CCC CA - #T CCG GAA GAT GAT GGA     462    Lys Leu Leu Ser Thr Met Ser Gly Ser Pro Hi - #s Pro Glu Asp Asp Gly    #            100    - GAA GGC AAT TGG ATA TGT CAG TAT ACG ACC AA - #A TCC AAA GAT ATT ATT     510    Glu Gly Asn Trp Ile Cys Gln Tyr Thr Thr Ly - #s Ser Lys Asp Ile Ile    #       115    - GCA TTT GCC ATA GAA GCA GAT GAT ATG CTT TC - #T CCA TTG AAA ATG ATG     558    Ala Phe Ala Ile Glu Ala Asp Asp Met Leu Se - #r Pro Leu Lys Met Met    #   130    - AGA TAT CTC AGA ACA ATC CGC TGATAAGTGG ATAACACCT - #G CACGGACACG     609    Arg Tyr Leu Arg Thr Ile Arg    135                 1 - #40    #653               CCGT GATTTAACGA ATAGTTAAGA GCTC    - (2) INFORMATION FOR SEQ ID NO:46:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 141 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    - Met Met Leu Lys Thr Gln Leu Thr Ala Phe Il - #e Gly Ala Val Ile Leu    #                 15    - Ala Gly Ser Ser Leu Ala Asn Pro Ile Lys Pr - #o Glu Val Cys Pro Ser    #             30    - Val Pro Ser Ile Gln Ser Glu Gly Met Ser Me - #t Ser Ser Glu Ile Leu    #         45    - Glu Gly Met Tyr Ile Thr Tyr Asn Leu Ser Hi - #s Tyr Asn Thr Ser Ser    #     60    - Ser Trp Val Phe Ile Val Gly Pro Ile Ala Al - #a Glu Asn Asp Asp Met    # 80    - Ala Leu Ala Glu Ser Asn Lys Leu Leu Ser Th - #r Met Ser Gly Ser Pro    #                 95    - His Pro Glu Asp Asp Gly Glu Gly Asn Trp Il - #e Cys Gln Tyr Thr Thr    #           110    - Lys Ser Lys Asp Ile Ile Ala Phe Ala Ile Gl - #u Ala Asp Asp Met Leu    #       125    - Ser Pro Leu Lys Met Met Arg Tyr Leu Arg Th - #r Ile Arg    #   140    - (2) INFORMATION FOR SEQ ID NO:47:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: other nucleic acid    #= "Primer"A) DESCRIPTION: /desc    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    # 19               ATC    __________________________________________________________________________

We claim:
 1. A frgA gene of Legionela pneumophila or a fragment thereof at least 18 basepairs in length.
 2. The gene of claim 1 which is the frgA gene of L. pneumophila strain 130b.
 3. A non-chromosome nucleic acid molecule having a sequence selected so that the nucleic acid molecule specially hybridizes under conditions of high stringency to a frgA gene or an mRNA encoded by a frgA gene.
 4. The molecule of claim 3 which is labeled.
 5. The molecule of claim 3 which has the sequence given in FIG. 7 (SEQ ID NO:4) or a fragment thereof.
 6. A kit for detecting or quantitating Legionella pneumophila comprising a container holding at least one nucleic acid molecule of claim
 3. 7. The kit of claim 6 wherein the molecule is labeled.
 8. The kit of claim 6 wherein the molecule has the sequence given in FIG. 7 (SEQ ID NO:4) or a fragment thereof.
 9. A method of detecting or quantitating Legionela pneumophila comprising:obtaining DNA from a sample suspected of containing L. pneumophila; amplifying the DNA by polymerase chain reaction using a plurality of nucleic acid molecules according to claim 3 as primers; and detecting or quantitating the L. pneumophila by detecting or quantitating the amplified DNA.
 10. The method of claim 9 wherein the nucleic acid molecules are labeled to allow for detection or quantitation of the amplified DNA.
 11. The method of claim 9 wherein labeled nucleotides are used during the polymerase chain reaction to allow for detection or quantitation of the amplified DNA.
 12. The method of claim 9 wherein a nucleic acid molecule of claim 7 which hybridizes to the amplified DNA is added after the polymerase chain reaction to allow for detection or quantitation of the amplified DNA.
 13. The method of claim 9 wherein the observation of a DNA of the expected size of the amplified DNA allows for detection or quantitation of the amplified DNA.
 14. A method of detecting or quantitating Legionela pneumophila comprising:obtaining DNA or RNA from a sample suspected of containing L. pneumophila; contacting the DNA or RNA with a nucleic acid molecule of claim 3 so that the molecule hybridizes to the DNA or RNA; and detecting or quantitating the L. pneumophila by detecting or quantitating the nucleic acid molecule hybridized to the DNA or RNA. 